Carbohydrate‐Rich Fraction of Aloe vera (L.) Burm.f. Extract Mitigates Bone Loss and Improves Metabolic Disturbance in Estrogen‐Deficient Rats

ABSTRACT

Aloe vera (L.) Burm.f., (AE) herb has been shown to have osteogenic, anti‐diabetic, and prebiotic activities in animal and human studies. Postmenopausal women generally exhibit massive bone loss, impaired intestinal calcium absorption, obesity‐related insulin resistance, and fat accumulation in the liver. It was possible that the AE herb may have a potential as a remedy for bone and metabolic disturbances associated with estrogen deficiency. Sham and ovariectomized rats were divided into 2 subgroups, that is, receiving daily administration of distilled water or 50 or 100 mg/kg of AE via either oral administration (p.o.) or intraperitoneal injection (i.p.) for 8 and 12 weeks. Nine weeks after ovariectomy, rats developed metabolic disturbances, as evidenced by obesity, impaired glucose tolerance, and high serum cholesterol levels. AE supplementation, either by p.o. or i.p., alleviated metabolic aberrations by improving glucose tolerance, reducing body weight, and decreasing fat deposition by increasing serum insulin levels. Furthermore, AE supplementation restored ovariectomy‐associated calcium malabsorption to that of sham. At week 12 post‐ovariectomy, massive bone loss was observed at trabecular‐rich regions. Daily AE supplementation at 50 mg/kg for 12 weeks, but not 8 weeks, significantly increased BMD and BMC compared with those of sham. Additionally, AE enhanced bone formation and suppressed bone resorption, as shown by bone histomorphometry and serum bone turnover markers. These findings clearly demonstrated the anti‐diabetic and osteogenic properties of Aloe vera extract in ovariectomized rats. Thus, Aloe vera had a potential as a nutraceutical candidate for the treatment of osteoporosis and metabolic disturbances associated with estrogen deficiency.

Unveiling Aloe vera's potential: A nutraceutical candidate to mitigate osteoporosis and metabolic aberrations linked to Estrogen deficiency.

Abbreviations

AE

Aloe vera extract

BMD

bone mineral density

BV/TV

bone volume fraction

Ct

cortical

Ct.A

cortical area

Ct. Sub

bone region including cortical shell and subcortical compartment

ES/BS

active eroded surface fraction

Es.Pm

endosteal perimeter

MC

bone mineral content

OAG

Aloe vera ‐treated ovariectomized rats (p.o.)

OAP

Aloe vera ‐treated ovariectomized rats (i.p.)

Ob.S/BS

osteoblast surface fraction

Oc.S/BS

osteoclast surface fraction

OV

ovariectomized rats

Ps.Pm

periosteal perimeter

SAG

Aloe vera ‐treated sham (p.o.)

SAP

Aloe vera ‐treated sham (i.p.)

Sp

separation

SV

sham‐operation

Tb

trabecular

Th

thickness

TOT

total tissue

1. Introduction

Osteoporosis is a systemic skeletal disease characterized by low bone mineral density and deterioration of bone microarchitecture, resulting in reduced bone strength and increased susceptibility to fractures. Osteoporosis is often referred to as the silent killer because patients are unaware of their bone loss until they experience fractures that lead to disability and eventually premature death. It is well known that estrogen deficiency is the major cause of osteoporosis in both animals and women. In addition to causing massive bone loss, estrogen deficiency is also associated with insulin resistance, dyslipidemia, lipid accumulation in the liver and impaired intestinal calcium absorption [1, 2, 3, 4]. The mechanisms underlying these metabolic disturbances in estrogen deficiency are not fully understood. Currently, there is no therapeutic treatment that addresses all aspects of menopause‐related pathological conditions (i.e., obesity, osteoporosis and cardiovascular disease).

Herbal medicines have been known to exhibit pharmacological and biological actions in the prevention and treatment of metabolic disturbances, potentially reducing the need for conventional drugs [5, 6]. Aloe vera has diverse health benefits and has traditionally been used to treat skin injuries such as burns, cuts, insect bites, eczemas, and digestive problems due to its anti‐inflammatory, antimicrobial, and wound‐healing properties. Aloe vera also displays antioxidant, anti‐diabetic, antihypertensive, and osteogenic properties [7]. Research on this medicinal plant has primarily focused on validating its traditional uses and understanding the mechanisms of action and identifying the active compounds. The most investigated active compounds are aloe‐emodin (1,8‐dihydroxy‐3‐hydroxyl‐methylanthraquinone), aloin, aloesin, and acemannan ((1,4)‐acetylated polymannose). Regarding effects on bone, Aloe vera crude extract has been shown to stimulate bone regeneration, while aloin has been found to inhibit osteoclastogenesis and stimulate osteoblastogenesis in an in vitro study [8]. Moreover, acemannan, a polysaccharide found in Aloe vera extract, has been reported to induce bone formation by stimulating bone stem cell proliferation, differentiation, and extracellular matrix synthesis [9]. However, studies on Aloe vera supplementation and bone remodeling in the in vivo osteoporosis model are limited. Several studies in rats [10, 11], mice [12, 13], and humans [14, 15] showed that crude Aloe vera extract supplementation alleviated metabolic disturbance in diabetic models. It was also shown that aloe‐emodin exhibited an inhibitory effect on the inflammatory response in the high‐fat induced cardiomyopathy by suppressing the pyroptosis pathway mediated by NLRP3 inflammasome [16]. The NLRP3 inflammasome, a multiple protein complex comprising nucleotide‐binding domain and leucine‐rich repeat protein 3, plays a key role in chronic low‐grade metabolic inflammation. Its excessive activation upregulated proinflammatory cytokines and induced insulin resistance in both rodents and humans [17, 18]. The NLRP3 inflammasome stimulated the maturation and secretion of IL‐1β, which promoted β cell dysfunction and pyroptosis (a programmed cell death triggered by inflammation). Also, IL‐1β impaired insulin signaling in the liver, muscle, and adipose tissue [19]. Another study on the effect of aloe‐emodin on microvascular dysfunction found that angiotensin II‐induced endothelial dysfunction could be alleviated by aloe‐emodin through NLRP3 inflammasome ubiquitination [20], suggesting potential therapeutic effects in early‐stage hypertension‐related cardiovascular disease. Furthermore, bone marrow cells of estrogen deficient rats showed upregulated protein expressions of NLRP3, caspase‐1 (a downstream protein of NLRP3) and IL‐1β all of which compromised osteoblast and osteoclast differentiation, possibly leading to bone loss [21]. Notably, the upregulation of those 3 proteins was reduced by estrogen replacement [22]. Additionally, it was hypothesized that acemannan should enhance intestinal calcium absorption since several in vitro and in vivo studies in rodents have shown that short‐chain fatty acids (produced by bacterial digestion of polysaccharides) facilitate intestinal calcium absorption [23, 24]. Therefore, Aloe vera extract could be a promising traditional medicine candidate for treating massive bone loss, metabolic disturbances, and calcium malabsorption in the estrogen deficient stage.

The ideal strategy for treating osteopenic or osteoporotic disorders is an inhibition of bone resorption with a concomitant increase in bone formation. Interestingly, bioactive ingredients in Aloe vera possess both anti‐osteoclastic and osteoblastogenetic activities. Up until now, there have been limited in vivo studies investigating the effects of Aloe vera supplementation on bone metabolism in estrogen deficiency‐induced osteoporosis animal models. Moreover, the information regarding the pharmacokinetics of bioactive components found in Aloe vera is limited, and the optimal route of Aloe vera supplementation needs to be investigated. The present study thus aims to examine the beneficial effect of Aloe vera supplementation derived from crude leaf extract and administered via oral gavage or intraperitoneal injection on the prevention of osteoporosis in an ovariectomy model.

2. Materials and Methods

2.1. Animals

Eight‐week‐old female WMN/Nrs Wistar rats weighing 200 ± 20 g, from the National Laboratory Animal Center, Salaya, Nakhon Pathom, Thailand, were acclimatized in a controlled environment of 25°C ± 2°C, on a 12:12‐h light–dark cycle with an average illumination of 200 lx for 7 days before the experiment. Animals were fed with rodent chow containing 1.0% w/w calcium, 0.70% w/w phosphorus, and 4,000 IU/kg 25(OH)D₃ (CP, Bangkok, Thailand) ad libitum with free access to reverse osmosis (RO) water. The present research was approved by the Animal Care and Use Committee, Faculty of Science, Mahidol University, Bangkok, Thailand (protocol number MUSC65‐020‐613). Central Animal Facility, Faculty of Science, Mahidol University has been internationally accredited by AAALAC. All rats were anesthetized by injection of 5 mg/kg xylazine (Thai Meiji Pharmaceutical Co. Ltd., Bangkok, Thailand) and 40 mg/kg zoletil (Virbac Laboratory 06516 Carros–France). Regarding euthanasia, rats were subjected to 15 mg/kg xylazine and 120 mg/kg zoletil injection for general anesthesia prior to cardiac removal. All experiments and methods used in the present study were performed in accordance with relevant regulations and the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guideline.

2.2. Experimental Design

The objectives of this experiment were to investigate the anti‐diabetic and osteogenic properties of Aloe vera extract (AE) in ovariectomized rats (OV). After one week of acclimatization, all rats were randomly allocated into sham operation and ovariectomy. In OV groups, rats underwent bilateral ovariectomy using a double dorso‐lateral approach under anesthesia with 2%–5% isoflurane. In the sham group, rats underwent bilateral laparotomy and the ovaries were only exposed but not removed. There were 2 sets of rats. In the first set, OV rats were supplemented with AE or distilled water once daily for 8 weeks. In the second set, rats received the similar treatments once daily for 12 weeks. For the first set, rats were randomly divided into sham‐operated (S) and ovariectomized (OV) groups. Sham‐operated rats were administered distilled water by p.o. and i.p. (SV). The OV group was further divided into 5 subgroups that were supplemented with (i) distilled water via p.o. and i.p. (OV control), (ii) p.o. AE 50 mg/kg (OAG50), (iii) p.o. AE 100 mg/kg (OAG100), (iv) i.p. AE 50 mg/kg (OAP50), and (v) i.p. AE 100 mg/kg (OAP100). For the second set, after one week of acclimatization, sham‐operated rats were subdivided into 3 subgroups that were supplemented with (i) distilled water via p.o. and i.p., (ii) p.o. AE 50 mg/kg (SAG50) and (iii) i.p. AE 50 mg/kg (SAP50). OV rats were subdivided into 4 groups that were (i) OV control, (ii) OAG50, (iii) OAG100, and (iv) OAP50. The experimental groups were shown in tabular style in Tables S1 and S2. The dosage and treatment duration used in the present study were based on the previous findings by Walid and co‐workers [10]. AE solution was freshly prepared and passed through a 0.2 μm sterile membrane filter before being administered to rats. Body weight was recorded on a weekly basis. An oral glucose tolerance test was performed at 5 and 9 weeks after surgery. Determination of intestinal fractional calcium absorption was performed after 8 weeks of AE treatment. At the end of the experiment, both sets of rats were fasted for 12 h before being euthanized. Intestinal segments were collected for investigating the expression of calcium transporter proteins by immunofluorescence. The liver and pancreas were collected for H&E staining, immunostaining against fibroblast growth factor‐21 and receptors (a glucose and lipid metabolism regulating hormone produced mainly in the liver), cell senescence markers (i.e., P16 and P21), proinflammatory cytokines, and proteins related to insulin signaling molecules. Gastrocnemius was also used for investigating the expression of insulin signaling molecules. Femora and the 5th lumbar vertebrae were removed from the carcass and cleaned of adhering tissues, wrapped in normal saline‐soaked gauze, and kept frozen at −20°C until analysis. Tibiae were subjected to bone microstructural analysis by bone histomorphometry. Serum was kept frozen at −80°C for chemical analyses.

2.3. Aloe vera Extract Preparation and Determination of Active Ingredients

The extraction procedure of Aloe vera was adapted from the report of Minjares‐Fuentes and co‐workers (2017). Briefly, fresh Aloe vera leaves supplied by Bangkok Aloe Ltd., Part. (Nakhon Prathom, Thailand) were washed with water. Aloe vera gel (raw material) was harvested from leaf pulp and carefully avoided latex and sap of leaves, which are known to cause health problems. Aloe gel was mixed well with reversed osmosis water in a ratio of 1:10 ( Aloe vera gel: water). The mixed solution was stirred at 60°C at a low speed of 400–500 rpm for 3 h and then centrifuged at 5000 × g at 4°C for 30 min. Thereafter, the clear supernatant was collected, mixed well with absolute ethanol in a ratio of 1:2, and left at 4°C overnight. On the next day, the cold solution was centrifuged at 5000 × g at 4°C for 30 min. The supernatant was discarded and the precipitation (water‐soluble fraction) was collected and re‐suspended in reversed osmosis water (1 mL of water per gram of raw material). The suspension was freeze‐dried into white powder and kept at 25°C until analysis. Aloe vera powder was re‐suspended in sterile distilled water and filtered through a 0.2 μm sterilizing filter membrane immediately before administration to animals.

2.4. Acemannan Fingerprint by ¹H Nuclear Magnetic Resonance (NMR)

¹H‐NMR analysis of the acemannan fingerprint was performed using the methodology adapted from Minjares‐Fuentes et al. (2017). A Bruker AVANCE III 500 MHz (Massachusetts, USA) probe head featuring an inverted triple resonance TXI (¹H, ¹³C, and ¹⁵N) was used to record the ¹H NMR spectra of the sample at 500 MHz. One milliliter of 99.9% deuterium oxide (Sigma‐Aldrich, Spain) was used to solubilize two micrograms of crude extract that had been weighed into a 2.0 mL tube. The sample was then solubilized and put into Wildman Economic 5 mm NMR tubes. Two milligrams of 99.5% tetramethylsilane standard (Sigma‐Aldrich, Spain) were added as an internal shift standard since the peaks corresponding to tetramethylsilane appear after 1.30 ppm, which were well‐separated from other Aloe‐derived proton peaks. ¹H‐NMR spectra of the acemannan fingerprint were shown in Figure S1. The NMR spectrum clearly showed the signals corresponding to the acetyl group of the acemannan molecule in the crude extract of Aloe vera . According to previous protocol [25] and [26], the acetyl groups of acemannan produced a distinctive signal (2.00–2.26 ppm) in the ¹H NMR spectrum, which can be interpreted as the fingerprint of acemannan composition in Aloe vera .

2.5. Determination of Monosaccharide Compositions and Total Polysaccharide

Monosaccharide compositions of AE were determined according to a previous protocol [27]. Briefly, the AE powder (1% w/v) was dissolved in 1 M H₂SO₄ and heated at 100°C for 2 h. Then, the solution was 100‐fold diluted with deionized water and filtered with a 0.45‐μm membrane filter to remove particulates. Fifty microliters of the diluted sample were injected into a high‐performance anion exchange chromatography with pulsed amperometric detection (HPAEC‐PAD) system (Dionex, Sunnyvale, CA) using a 250 mm × 4.0 mm (CaboPac PA1) column. The mobile phases were 100 mM NaOH (A) and deionized water (B) and ran over 45 min at a flow rate of 0.4 mL/min. The chromatograms were obtained. The content of notable monosaccharides (i.e., arabinose, galactose, glucose and mannose) was calculated in mg/L. Monosaccharide standards (i.e., arabinose, galactose, glucose and mannose) were purchased from Sigma Aldrich.

Total amount of polysaccharide was analyzed based on previous method [28]. Briefly, 10 mg of AE powder was dissolved in 100 mL of deionized water. Thereafter, 1 mL of the prepared solution was mixed with 1 mL of 5% phenol and 5 mL of 95% H₂SO₄. The mixture was incubated at room temperature for 10 min, and the optical density was measured at 490 nm to determine total amount of polysaccharide. The analysis was conducted in three replicates.

Monosaccharide compositions and total amount of polysaccharide were shown in Table S3. The total amount of polysaccharide of AE powder was 91.17 ± 1.28 g/100 g, suggesting the extraction protocol used in the present study yielded a carbohydrate‐rich fraction. The solubility test was shown in Table S4.

2.6. Determination of Serum Calcium, Inorganic Phosphate, and Lipid Profiles

Whole blood was allowed to clot at room temperature for 1 h, then centrifuged (model D‐37520; Kendro Laboratory Products, Hanau, Germany) at 1500 × g for 10 min at 4°C. Clotted blood samples were used to determine total serum calcium and serum inorganic phosphate by the o‐cresolphthalein complexone method (model Dimension RxL analyzer, Dade Behring, Marburg, Germany). The lipid profile was analyzed by enzymatic colorimetric assay at Ramathibodi Hospital laboratory, Bangkok, Thailand. To measure serum triglyceride, lipase/glycerol kinase/glycerol‐3‐phosphate oxidase were used, and cholesterol was determined by using the cholesterol oxidase enzyme. The enzymatic method is widely used to determine lipid profile in mammalian samples.

2.7. Determination of Serum Levels of Bone Formation and Bone Resorption Markers, Fibroblast Growth Factor‐21, Insulin, 1,25(OH) ₂D₃ and 17‐β Estradiol

Sera were thawed at room temperature prior to the determination of bone turnover markers by using commercial ELISA kits, that is, procollagen type 1 amino‐terminal propeptide; P1NP, a bone formation marker (AC‐33F1, Immunodiagnostic Systems, UK) and tartrate‐resistant acid phosphatase; TRAP‐5b, a bone resorption marker (SB‐TR102, Immunodiagnostic Systems, UK), glucose regulating hormones (i.e., insulin (EZRMI‐13 K, Sigma Aldrich, USA) and fibroblast‐growth factor 21 (ab223589, abcam, UK)), 1,25(OH)₂D₃ (E‐EL‐0016, Elabscience, Texas, United State) and 17‐β estradiol (ab108667). The protocols were conducted according to the manufacturer's instructions.

2.8. Determination of Intrahepatic Triglyceride and Lipid Peroxidation (Malondialdehyde; MDA)

Intrahepatic triglyceride and MDA were determined by commercial colorimetric assay kits (Cat# ab65336 and ab118970, respectively) according to the manufacturer's protocol. Approximately 100 mg of liver was homogenized in the provided lysis buffer and centrifuged; the supernatant was analyzed for triglyceride content. Hepatic triglyceride content was expressed as nmol/mg tissue. As for MDA determination, liver homogenate was centrifuged at 13000 × g at 4°C for 10 min, and the supernatant was used to determine MDA. Hepatic MDA content was expressed as nmol/mg liver tissue.

2.9. Determination of mRNA Expression of Gastrocnemius and Liver by RT‐PCR

Total RNA of gastrocnemius and liver was extracted using TRIzol reagent (Invitrogen, Carlsbad, United States) and the RNA concentration was quantitated by NanoDrop‐2000c spectrophotometer (Thermo Specific, Waltham, MA, USA). Then, RNA was reverse‐transcribed into cDNA by iScript cDNA Synthesis Kit (Bio‐Rad Laboratories, CA, United State). Quantitative real‐time PCR was performed by QuantStudio 3 Real‐time PCR system (Applied Biosystems, MA, United State). Changes in mRNA expression were normalized by β‐actin (ACTB) and were presented as relative fold changes to sham‐operation control. The sequences of specific primers were shown in Table S5.

2.10. Determination of Intestinal Calcium Transporter Protein Expression by Immunofluorescence, and GIP/GLP‐1 by Immunoperoxidase Staining

Duodenal, ileal, and cecal segments were cleaned of luminal content by flushing with chilled 0.1 M phosphate‐buffered saline (PBS) and fixed overnight in PBS containing 4% paraformaldehyde. Tissues were dehydrated in graded alcohol and embedded in paraffin; then the specimens were cut longitudinally into 4‐μm sections, which were later subjected to antigen retrieval as described in Table S6. Non‐specific binding was blocked for 2 h with 10% normal goat serum and 0.1% Tween‐20 in PBS. Thereafter, sections were incubated at 4°C overnight in a moist chamber with primary antibodies. As for immunofluorescence, sections were washed 3 × 10‐min with 0.5% Tween‐20 in PBS before being incubated for 1 h at room temperature in the dark with anti‐rabbit Dylight 594 (Cat#DI‐1594, Vector Laboratories), anti‐rabbit Alexa Fluor 488 (ab150077, Abcam, Cambridge, UK) or anti‐mouse Alexa Fluor 594 (Cat#150116, Abcam) in blocking buffer. Sections were mounted with slow fade Diamond antifade mounting medium containing DAPI (Cat# S36964, Invitrogen), visualized under a fluorescent microscope (model eclipse Ni‐U; Nikon) and a confocal laser‐scanning microscope with AriScan (model, Zeiss LSM800; Carl Zeiss AG, Germany) equipped with Zeiss Zen Blue version 2.1 Software. For immunoperoxidase staining, sections were incubated for 1 h at room temperature with anti‐rabbit conjugated with biotin (Cat# 31820, Invitrogen), washed, and then incubated for 1 h with Streptavidin HRP conjugate (Cat# 434323, Invitrogen). The color was developed with the DAB substrate chromogen system (Cat# K3468, Dako).

2.11. Determination of Protein Expression of Liver, Pancreas, Gastrocnemius, Ileum, Cecum by Western Blot

Ten milligrams of tissue samples (i.e., liver, pancreas, gastrocnemius, ileum, cecum) were homogenized in 0.5 mL of radioimmunoprecipitation assay (RIPA) buffer for 1 h, then centrifuged for 10 min at 12000 rpm. Protein concentrations were determined using the bicinchoninic acid protein assay (Pierce Biotechnology, IL, United State). Protein samples were denatured at 95°C for 5 min, then the samples (40 μg/well) were separated by 10%–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to a nitrocellulose membrane. The membranes were blocked with 4% bovine albumin for 1 h and were incubated at 4°C with specific primary antibodies overnight (Table S7). After washing, membranes were incubated with 1:1000 goat anti‐rabbit IgG horseradish peroxidase (HRP)‐linked antibody (Cat# 7074S; Cell Signaling, Danvers, MA, USA) or 1:1000 goat anti‐mouse‐HRP antibody (Cat#ab6789, Abcam) and signals were developed by using enhanced chemiluminescent (ECL) horseradish peroxidase substrate (Thermo Fisher Scientific, United State). Levels of protein expression were determined in triplicate, and their intensities were quantified by ImageJ 1.50i software (National Institute of Health, USA). Mean and variance of data were displayed in respective bar graphs.

2.12. Bone Histomorphometry

Tibiae were cleaned of adhering muscle and cut into 2 cm length pieces. After bone marrow was flushed with 0.9% normal saline, tibiae were dehydrated and embedded in methyl methacrylate resin in a posterior end down position. Bone specimens were then incubated at 42°C to allow the resin to become fully polymerized; they were then kept at 4°C until use. Prior to analyses, bone specimens were longitudinally cut into 7‐μm‐thick sections with a microtome equipped with a tungsten carbide knife (model RM2265; Leica, Nussloch, Germany). Bone sections were then mounted on gelatin‐coated microscope slides, deplastinated, dehydrated, and stained with Goldner's trichrome. Bone microarchitecture was analyzed under a light microscope using the computer‐assisted Osteomeasure system (Osteometric Inc., Atlanta, GA, USA), operated with software version 4.1. The region of interest was located at secondary spongiosa, that is, 2–4 mm distal to the epiphyseal plate. Parameters were trabecular bone volume normalized by tissue volume (bone volume fraction; BV/TV, %), trabecular thickness (Tb.Th, μm), trabecular separation (Tb.Sp, μm), trabecular number (Tb.N, mm⁻¹), osteoblast surface normalized by bone surface (Ob.S/BS, %), osteoclast surface (Oc.S/BS, %) and active eroded surface (aES/BS, %).

2.13. Bone Mineral Density and Mineral Content, and 3‐Dimension Microstructural Analysis by Peripheral Quantitative Computed Tomography and Micro‐Computed Tomography

After routine calibration with the standard phantom supplied by the manufacturer, femora were scanned using peripheral Quantitative Computed Tomography (pQCT) in the research M mode (XCT Research SA+, Stratec Medizintechnik GmbH., Germany) with a 50 kV X‐ray tube, a current of 0.307 mA, and a voxel size of 0.1 mm³, and were analyzed using XCT‐5.50E software (Stratec, Medizintechnik GmbH., Germany). Regions of interest were at the distal metaphyseal femur and mid‐shaft diaphyseal femur. The femur was segmented into 3 compartments based on density, that is, cortical shell, subcortical, and trabecular compartments. Measured parameters were bone mineral density (BMD), bone mineral content (BMC) and cortical bone structure. Total content (TOT.BMC) was defined as bone content including all 3 compartments mentioned above, and Ct.Sub.BMC was defined as the bone content of the cortical shell and subcortical compartment. Cortical microstructure parameters were cortical thickness (Ct.Th), cortical area (Ct.A), cortical periosteal perimeter (Ct.Ps.Pm), and endosteal perimeter (Ct.Es.Pm). As for the metaphyseal region, measured parameters were averaged from three points of volume of interests (VOI) that were at 2, 3, and 4 mm away from the distal growth plate, while for the diaphyseal region, parameters came from a single measurement at cortical mid‐shaft.

With higher resolution instruments, femur and 5th lumbar vertebrae were analyzed by ultra‐high resolution micro‐computed tomography (model UHR U‐CT, Milabs, Utrecht, Netherlands). Instruments were routinely calibrated with 5 points HA phantoms (i.e., 1200, 800, 200, 50 and 0 mg HA/cm³; model QRM microCT‐HA D25) before scanning bone specimens. As for the femur, the region of interest was located at secondary spongiosa, that is, 2.0–4.0 mm below the growth plate. Meanwhile, in lumbar vertebrae, the region of interest was 0.5–1.5 mm below the growth plate. The parameters were trabecular BMD, trabecular BMC, bone volume normalized by tissue volume (BV/TV, %), trabecular thickness (Tb.Th, mm), and trabecular separation (Tb.Sp, mm) analyzed by Imalytics Preclinical software (version 13.0).

2.14. Calcium Determination in Feces by Flame Atomic Absorption Spectrophotometry

Fractional intestinal calcium absorption (FCa) was determined in 8‐week AE‐supplemented rats. FCa was averaged from 3 experimental days by collecting the fecal pellets while rats were housed in individual metabolic cages (Techniplast, Venice, Italy). Fecal pellets were dried at 80°C for 3 days and then ashed at 800°C overnight in a muffle furnace (model 48 000; Thermolyne, Dubuque, IA). Fecal dry and ash weights were recorded. Fecal ash (1 mg) was dissolved in an acid solution (i.e., a mixture of 65% v/v HNO₃ and 37% v/v HCl in ratio of 8:1) and stirred until completely dissolved. Fecal calcium contents were determined by flame atomic absorption spectrometry (PerkinElmer, MA, USA).

3. Statistical Analysis

Results were expressed as means ± SE. The type I error (alpha) was set at 0.05 for all statistical analyses. The difference between two groups was determined by unpaired Student t‐test (i.e., 17β estradiol level for sham vs. ovariectomy). One‐way analysis of variance with Tukey multiple comparisons test was used for multiple sets of independent data. The interaction of AE extract supplementation and ovariectomy was analyzed by two‐way analysis of variance with multiple comparisons. The level of significance for statistical test was p < 0.05. All data were analyzed by GraphPad Prism 10 for macOS (GraphPad Software, San Diego, CA).

4. Results

4.1. The Removal of Ovaries for 9 Weeks Impaired Glucose and Lipid Metabolism. Aloe vera Extract Supplementation Alleviated Insulin Resistance But Did Not Correct Dyslipidemia

Serum 17β‐estradiol levels were significantly lower in ovariectomized rats (OV) compared to sham operated rats (SV) (i.e., 25.01 ± 4.77 pg/mL in SV vs. 7.50 ± 1.73 pg/mL in OV, p < 0.01), confirming ovariectomy. There were no differences in the weight of major organs (i.e., liver, kidney, spleen and heart) between the non‐AE treated and the AE‐treated OV rats (Table 1). From H&E staining of liver (Figure S2), there were no signs of tissue lesions, inflamed regions, or tissue necrosis suggesting that AE administration at the applied dosage was unlikely to cause toxicity. The differences in organ weight normalized by body weight between SV and OV groups were due to the higher body weight in OV rats, which exhibited body weight gain as early as the second week after ovariectomy (Figure 1a). In addition, OV rats showed higher food intake and increased fat depots at urogenital and perirenal fat (Table 1). Intraperitoneal injection of 50 mg/kg AE in OV rats (OAP50) reduced weight gain starting in the 4th week of treatment, while AE given orally showed a delayed effect, that is, starting in the 9th week of treatment (Table S8). The reductions in weight gain and fat deposition were more dramatic in the 100 mg/kg OAG group compared to those receiving 50 mg/kg (Figure 1a and Table 1); therefore, we chose the dose of 100 mg/kg to investigate the antidiabetic properties of AE. An oral glucose tolerance test showed that 9 weeks after ovariectomy, OV rats exhibited impaired glucose tolerance with a significantly higher HbA1C level and higher serum insulin levels compared to the sham group (Figure 1b,c and Table 2); meanwhile, these metabolic disturbances were not observed in 5 weeks post‐surgery (Figure S3). OV rats down‐regulated phosphorylated IRS1/Akt while up‐regulating total Akt protein in the liver and gastrocnemius, as well as down‐regulating mRNA expression of IRS‐1 in the gastrocnemius. Since Akt and IRS‐1 are downstream target proteins of insulin, the results suggested that insulin resistance developed in OV rats (Figure 1d–f; Figures S4 and S5a). Impaired glucose homeostasis in OV rats was restored to SV levels by 100 mg/kg AE in both OAG and OAP (Figure 1d,e; Figures S4 and S5a). Serum levels of insulin were significantly increased in OAG and increased further in OAP (Table 2) resulting in a reversal of the protein expression levels of p‐IRS1, p‐Akt, total Akt, and mRNA expression of IRS‐1 and GLUT4 to SV levels (Figure 1d,e; Figures S4 and S5a). Hyperglycemia with hyperinsulinemia in OV rats resulted in higher protein expression of glucagon‐like peptide in the ileum, which is known to stimulate insulin secretion. This glucagon‐like peptide was restored to SV levels in AE‐treated OV rats (Figure 1e; Figures S5b and S6), suggesting that hyperinsulinemia in OAG and OAP was not the result of incretin stimulation.

TABLE 1.

Organ weight, blood chemistry and lipid profiles. Rats were divided into non‐treated sham operation (SV) and ovariectomy (OV). OV rats received distilled water (OV control) or Aloe vera extract by oral administration (OAG) or intraperitoneal injection (OAP) in the dose of 50 or 100 mg/kg, once daily for 8 weeks.

	SV (n = 5)	OV (n = 5)	100 mg/kg AE		50 mg/kg AE
			OAG (n = 5)	OAP (n = 5)	OAG (n = 5)	OAP (n = 5)
Food intake (g/day)	14.95 ± 0.32	18.33 ± 0.62*	17.03 ± 0.99	15.78 ± 0.48	15.34 ± 0.82 †	16.00 ± 0.40
Organ weight (g/100 g BW)
Urogenital fat	0.48 ± 0.05	1.08 ± 0.07*	0.53 ± 0.06 †	0.49 ± 0.06 †	0.55 ± 0.02 †	0.73 ± 0.03*, †
Perirenal fat	0.13 ± 0.01	0.25 ± 0.04*	0.18 ± 0.01	0.17 ± 0.03	0.27 ± 0.03*	0.34 ± 0.02*
Liver	2.64 ± 0.09	2.18 ± 0.04*	2.24 ± 0.10*	2.22 ± 0.07*	2.37 ± 0.10	2.23 ± 0.06*
Kidney	0.62 ± 0.02	0.51 ± 0.01*	0.55 ± 0.02	0.53 ± 0.02*	0.58 ± 0.02	0.56 ± 0.02
Spleen	0.33 ± 0.01	0.29 ± 0.01*	0.32 ± 0.01	0.31 ± 0.01	0.33 ± 0.01	0.30 ± 0.01
Heart	0.36 ± 0.01	0.31 ± 0.01*	0.33 ± 0.01	0.33 ± 0.01	0.32 ± 0.01*	0.30 ± 0.01*
Uterine horns	0.28 ± 0.02	0.10 ± 0.01*	0.12 ± 0.03*	0.12 ± 0.04*	0.08 ± 0.01*	0.09 ± 0.01*
Organ weight (absolute, g)
Urogenital fat	1.14 ± 0.12	3.15 ± 0.26*	1.57 ± 0.23 †	1.43 ± 0.22 †	1.61 ± 0.04 †	2.35 ± 0.15*
Perirenal fat	0.32 ± 0.02	0.73 ± 0.12*	0.52 ± 0.02	0.50 ± 0.11	0.79 ± 0.08*	1.09 ± 0.08*, †
Liver	6.37 ± 0.42	6.29 ± 0.09	6.46 ± 0.06	6.32 ± 0.26	7.00 ± 0.14	7.17 ± 0.27
Kidney	1.48 ± 0.08	1.47 ± 0.04	1.59 ± 0.02	1.50 ± 0.06	1.71 ± 0.03	1.80 ± 0.07*, †
Spleen	0.80 ± 0.01	0.85 ± 0.03	0.93 ± 0.01	0.89 ± 0.06	0.98 ± 0.01*	0.96 ± 0.03*
Heart	0.87 ± 0.03	0.91 ± 0.03	0.97 ± 0.02	0.93 ± 0.06	0.95 ± 0.03	0.96 ± 0.02
Uterine horns	0.66 ± 0.05	0.28 ± 0.03*	0.33 ± 0.08*	0.31 ± 0.06*	0.25 ± 0.03*	0.29 ± 0.03*
Serum
TG (mg/dL)	20.50 ± 2.33	22.40 ± 2.31	22.00 ± 2.34	19.20 ± 1.53	N/A	N/A
TC (mg/dL)	51.00 ± 1.41	70.40 ± 3.00*	71.20 ± 4.22	69.20 ± 3.97	N/A	N/A
HDL (mg/dL)	21.00 ± 0.41	26.60 ± 0.81*	25.00 ± 1.05	24.40 ± 0.67	N/A	N/A
LDL (mg/dL)	2.50 ± 0.29	3.20 ± 0.37	3.80 ± 0.20	3.40 ± 0.40	N/A	N/A
non‐HDL (mg/dL)	30.00 ± 1.08	43.80 ± 2.17*	46.20 ± 3.45	44.80 ± 3.32	N/A	N/A
TC/HDL ratio	2.43 ± 0.05	2.64 ± 0.02	2.84 ± 0.11	2.82 ± 0.09	N/A	N/A
ALP (U/L)	56.25 ± 4.21	76.80 ± 3.25*	67.80 ± 2.20	62.80 ± 5.61	N/A	N/A

Note: Data was expressed as mean ± SE. The difference between groups was analyzed by one‐way ANOVA with Tukey's multiple comparison test.

^* p < 0.05 compared with SV group.

^† p < 0.05 compared with OV group.

FIGURE 1.

The effect of supplementation of Aloe vera extract (AE) on glucose and lipid metabolism in sham and ovariectomized rats. Sham (S) and ovariectomized (O) rats were supplemented with distilled water (SV and OV) or AE. For AE supplemented groups, rats were administered 50 mg/kg or 100 mg/kg via either oral gavage (SAG and OAG, n = 5 each) or intraperitoneal injection (SAP and OAP). Rats were sacrificed at 8 weeks and 12 weeks post‐supplementation. (a) percent body weight gain, figures B to P showed data from 8 weeks post supplementation, (b, c) oral glucose tolerance test and area under curve, (d, e) protein expression of phosphorylated Akt and total Akt in liver and pancreas by western blot analysis, (f) protein expression of GLP‐1 in ileum by western blot analysis, (g) Representative images of immunofluorescent stained against FGF‐21 in liver, (h) protein expression of FGF‐21 and its receptor FGFR‐1 and 3, phosphorylated AMPK, total AMPK and SirT‐1 in liver by western blot analysis, (i) H&E stained liver sections, arrows indicated triglyceride droplets. Whole blot with molecular marker was shown in Supplemental information (uncropped Western blot images). Results are expressed as means ± SE with individual values. The differences between experimental groups were determined by one‐way ANOVA followed by Tukey post hoc test. *p < 0.05 compared with SV, ^† p < 0.05 compared with OV.

TABLE 2.

Parameters related to glucose/lipid metabolism, and calcium and bone metabolism.

Parameters	SV (n = 5)	OV (n = 5)	OAG (n = 5)	OAP (n = 5)
Glucose and lipid metabolism
HbA1C (%)	4.08 ± 0.02	4.30 ± 0.06*	4.18 ± 0.03	4.24 ± 0.04
Serum insulin (ng/mL)	1.69 ± 0.11	2.97 ± 0.13*	5.98 ± 0.28* , †	7.12 ± 0.52* , † , #
Serum FGF‐21 (pg/mL)	162.8 ± 11.8	533.4 ± 26.9*	428.3 ± 16.8* , †	413.8 ± 15.7* , †
Liver triglyceride (nmole/mg)	9.54 ± 0.2	32.0 ± 1.2*	20.0 ± 1.1* , †	20.1 ± 1.4* , †
Calcium and bone metabolism
Serum 1,25(OH)2D3 (ng/mL)	60.5 ± 7.4	27.2 ± 3.1*	86.3 ± 8.1* , †	82.4 ± 11.8* , †
Serum calcium (mg/dL)	9.98 ± 0.09	10.00 ± 0.04	10.10 ± 0.04	9.80 ± 0.16
Serum inorganic phosphate (mg/dL)	4.47 ± 0.17	4.62 ± 0.27	4.54 ± 0.48	5.00 ± 0.28

Note: Rats were divided into non‐treated sham operation (SV) and ovariectomy (OV). OV rats were received distilled water (OV control) or Aloe vera extract by oral administration (OAG) or intraperitoneal injection (OAP) at 100 mg/kg, once daily for 8 weeks. Data was expressed as mean ± SE. The difference between groups was analyzed by one‐way ANOVA with Tukey's multiple comparison test.

^* p < 0.05 compared with SV group.

^† p < 0.05 compared with OV group.

^# p < 0.05 compared with OAG.

Dysregulation of lipid metabolism was observed in OV rats. They exhibited higher circulating levels of fibroblast growth factor‐21, a hormone regulating glucose and lipid metabolism (Table 2). Immunostaining and Western blot analyses showed that the liver, one of the major sources of FGF‐21 production, and its target organ in OV rats exhibited higher intensity of protein expression of FGF‐21 and its receptors (FGFR‐1 and 3), and exhibited lower intensity of p‐AMPK and SirT‐1, which are known downstream target molecules of FGF‐21 (Figure 1g,h and Figure S5c). OV rats also showed fat accumulation in the liver and elevated serum levels of cholesterol (i.e., total cholesterol, HDL and non‐HDL), TC/HDL ratio, and alkaline phosphatase, the latter of which suggested impaired liver function (Figure 1i and Table 1). AE supplementation in OV rats decreased serum levels of FGF‐21 and its expression intensity as well as that of its receptors (FGFR‐1 and 3) in the liver, but the serum levels in OAG and OAP rats were still significantly higher than in SV rats (Figure 1h; Figure S5c; Table 2). The change in liver triglyceride corresponded with the change in serum levels of FGF‐21 (Table 2). Unfortunately, AE supplementation for 8 weeks failed to ameliorate the OV‐associated dyslipidemia (Table 1). Moreover, prolonged AE administration for 12 weeks in OV rats did not improve hypercholesterolemia, even though it showed a tendency to reduce triglyceride (Figure S7). AE supplementation had no effect on serum calcium and inorganic phosphate (Table 2).

4.2. The Liver of Estrogen Deficient Rats Showed Signs of Oxidative Stress and an Increase in Pro‐Inflammatory Cytokines. AE Supplementation Alleviated Oxidative Stress and Restored the Levels of Proinflammatory Cytokines to Those of the Sham Group

Estrogen‐deficient OV rats exhibited higher lipid peroxidation (MDA, an oxidative stress marker) in the liver, leading to an increase in NLRP3 inflammasomes (Figure 2a–c). The overactivation of the NLRP3 inflammasome in OV rats, in turn, resulted in the overproduction of IL‐1β and IL‐6. AE supplementation at 100 mg/kg for 8 weeks significantly reduced oxidative stress and restored the expressions of IL‐1β and IL‐6 in OAG and OAP to the level of the sham group (Figure 2a–c), the MDA level in AE‐treated rats remained significantly higher than in the SV group. Furthermore, immunostaining of hepatic P21, a cell senescence marker, showed higher intensity in OV rats, with expression in OAG and OAP rats being restored to a level similar to those in SV rats, while the change in P16 showed a change in the opposite direction (Figure 2d–f). Similar changes in inflammasome and proinflammatory cytokines were observed in the pancreas (Figure 2g,h).

FIGURE 2.

Eight‐week supplementation with 100 mg/kg Aloe vera extract decreased pro‐inflammatory cytokines in estrogen‐deficiency. (a) malondialdehyde level in liver (markers for oxidative stress), (b, c) protein expression of NLRP3 inflammasome and pro‐inflammatory cytokines (i.e., interleukin‐1β and interleukin‐6) in liver by western blot analysis, (d, e) protein expression of P16 and P21, cell senescence markers by western blot analysis, (f) representative immunostaining against P16, (g, h) protein expression of NLRP3 inflammasome and pro‐inflammatory cytokines (i.e., interleukin‐1β and interleukin‐6) in pancreas by western blot analysis. Results are expressed as means ± SE with individual values. The differences between experimental groups were determined by one‐way ANOVA followed by Tukey post hoc test. *p < 0.05 compared with SV, ^† p < 0.05 compared with OV.

4.3. Estrogen Deficiency Impaired Intestinal Calcium Absorption. Oral Aloe vera Supplementation for 8 Weeks Alleviated This Impairment

Representative immunostaining images of TRPV6 (an apical calcium transporter channel), PMCA1 (a basolateral calcium transporter channel), Cldn‐5 (a selective cation channel located at tight junction) and zonula occludens‐1 (ZO‐1) in the duodenum and cecum were shown in Figure S8. Compared to SV rats, the duodenum of OV rats showed significant reductions in protein expression of TRPV6, PMCA1, Cldn‐5, and ZO‐1. AE administration by both routes in OV rats completely restored the expression of all four proteins to the sham levels. The change in protein expression in the cecum was similar to that in the duodenum (Figure S8). Moreover, oral administration of AE in the sham group (SAG) increased the intensity of calcium transporter protein expression compared to non‐treated SV rats. Protein expressions of calcium transporters and tight junctions in cecal specimens, as demonstrated by Western blot, confirmed the findings observed in the immunostaining experiment (Figure 3a,b). Regarding the calcium balance study (Figure 3c), fractional intestinal calcium absorption (FCa) was decreased in OV rats (67.92% ± 6.44%) but was restored after daily AE supplementation for 8 weeks at a dose of 50 mg/kg, with AE administration by the oral route being more effective than by intraperitoneal injection (117.8% ± 3.54% in OAG vs. 94.71% ± 2.34% in OAP, p value = 0.0132). FCa did not further increase when OAG rats received 100 mg/kg AE (99.63% ± 3.04%) (data not shown). Meanwhile, FCa was slightly increased, but not significantly, in the AE‐supplemented sham group. The reduction in circulating 1,25(OH)₂D₃ was similarly restored by AE supplementation (Table 2). It was likely that the reduction in fractional intestinal calcium absorption in OV rats was at least in part due to the lower circulating 1,25(OH)₂D₃ and decreased intestinal integrity. AE administration alleviated these impairments, that is, restoring protein expression of tight junction proteins and raising the circulating level of 1,25(OH)₂D₃ to the level found in the sham control, which in turn led to upregulation of calcium transporters in the small and large intestine.

FIGURE 3.

Eight‐week supplementation of 50 and 100 mg/kg Aloe vera extract increased protein expression of calcium transporters in small and large intestine, and increased fractional intestinal calcium absorption. Rats were supplemented with distilled water or 100 mg/kg Aloe vera extract for 8 weeks. (a, b) calcium transporter protein expression in cecum, and (c) fractional intestinal calcium absorption of 50 mg/kg AE supplemented rats. Results are expressed as means ± SE with individual values. The differences between experimental groups were determined by one‐way ANOVA followed by Tukey post hoc test. *p < 0.05 compared with SV, ^† p < 0.05 compared with OV, ^# p < 0.05 compared with AE‐treated OV rats p.o. (OAG).

4.4. Ovariectomized Rats Exhibited Bone Loss Primarily at the Trabecular‐Rich Region. AE Supplementation by Intraperitoneal Injection and Oral Administration Alleviated OV‐Induced Bone Loss in the Distal Femur and L5 Lumbar Vertebrae

Figure 4A showed the effect of daily 50 mg/kg AE supplementation by intraperitoneal injection (OAP) over 8 and 12 weeks on femora BMD, BMC, and bone microstructure. Intraperitoneal administration of AE to the OV rats for 12 weeks, but not 8 weeks, significantly increased Tb.BMD, Tb.BMC, Ct.Sub.BMC, Ps.Pm, and Es.Pm compared to corresponding parameters in non‐treated age‐matched OV rats (Figure 4b). Regarding the cortical compartment, 8 and 12 week‐AE supplementation in OAP significantly decreased Ct.BMD and Ct.Sub.BMD with no change in TOT.BMD, TOT.BMC, Ct.Th, and Ct.A (Figure 4b). Figure 4c showed the effect of AE on diaphyseal mid shaft femur. While 12 weeks of AE supplementation significantly decreased Ct.BMD, both 8 and 12 weeks of AE supplementation were found to significantly decrease Ct.BMC, Ct.Th, and Ct.A. There were no changes in Ps.Pm or Es.Pm at mid‐shaft femur (Figure 4c). The results from Figure 4 indicated that the effective duration of AE supplementation was 12 weeks; therefore, we selected 12 weeks of treatment for the subsequent experiments.

FIGURE 4.

Aloe vera extract supplementation for 12 weeks alleviated ovariectomy‐induced bone loss examined by pQCT. Ovariectomized rats were either intraperitoneal injected with 50 mg/kg Aloe vera extract (OAP) or distilled water (OV). Rats were sacrificed at 8 weeks and 12 weeks post‐supplementation. (a) experimental timeline, (b) pQCT analysis at distal metaphyseal femur, (c) pQCT analysis at mid‐shaft diaphyseal femur. A, bone area; BMC, bone mineral content; BMD, bone mineral density; Ct, cortical; Ct.Sub, bone region included cortical and subcortical compartment; Es.Pm, endosteal perimeter; Ps.Pm, periosteal perimeter; Tb, trabecular; Th, thickness; TOT, total tissue which included both trabecular and cortical compartments. Results are expressed as means ± SE with individual values. The difference between two set of data was determined by un‐paired student t‐test. *p < 0.05, **p < 0.01, ***p < 0.001 compared with corresponded age‐matched OV.

To investigate the effective route of administration, AE was daily administered to sham and ovariectomized rats by either p.o. or i.p. injection in the dose of 50 mg/kg for 12 weeks. The experimental plan was depicted in Figure 5a. Compared with the sham group, non‐treated OV rats exhibited bone loss primarily at the trabecular metaphyseal femur, as shown by a reduction in distal metaphyseal Tb.BMD, TOT.BMD, and Ct.Th, with increases in Tb.BMC, Ct.Ps.Pm, and Ct.Es.Pm, with no change in TOT.BMC, Ct.Sub.BMD, Ct.Sub.BMC, Ct.BMD, Ct.BMC, and Ct.A (Figure 5b). The mid‐shaft diaphyseal of non‐treated OV rats exhibited higher Ct.BMC, Ps.Pm, and Ct.A when compared with non‐treated sham (Figure 6). In the sham group, neither p.o. nor i.p. AE administration (SAG and SAP, respectively) had any effect on the distal metaphyseal or mid‐shaft diaphyseal femur (Figures 5b and 6, respectively). In contrast, AE administration in OV rats by both routes significantly increased Tb.BMD, while only i.p. AE‐treated OV rats exhibited a reduction in Ct.Sub.BMD and Ct.BMD. There was no change in other parameters in the metaphyseal (Figure 5b) and diaphyseal femur (except for a reduction in Ct.BMC in i.p. AE‐treated OAP rats) (Figure 6). Representative 3D micro‐CT images (frontal plane) of the femur in all treatments were depicted in Figure 5c. Besides bone loss, estrogen deficiency led to an increased linear bone growth, i.e., an increase in femoral length without any effect on mechanical properties. AE administration in both sham and OV rats did not have an effect on bone mechanical properties (Table 3).

FIGURE 5.

Aloe vera extract supplementation in the dose of 50 mg/kg, for 12 weeks, either by oral gavage or intraperitoneal injection similarly alleviated ovariectomy‐induced bone loss examined by pQCT. Sham and ovariectomized rats received either distilled water (SV and OV) or Aloe vera extract (AE) administration. For AE supplemented groups, rats were administered in the dose of 50 mg/kg either by oral gavage (SAG and OAG, n = 6 each) or by intraperitoneal injection (SAP and OAP). Rats were sacrificed at 12 weeks post‐supplementation. (a) experimental timeline, (b) pQCT analysis at distal metaphyseal femur and (c) representative 3D micro‐CT images of the femur. Scale bar, 1 mm. A, bone area; BMC, bone mineral content; BMD, bone mineral density; Ct, cortical; Ct.Sub, bone region included cortical and subcortical compartment; Es.Pm, endosteal perimeter; Ps.Pm, periosteal perimeter; Tb, trabecular; Th, thickness; TOT, total tissue which included both trabecular and cortical compartments. Results are expressed as means ± SE with individual values. Two‐way ANOVA followed by Tukey's multiple comparisons post hoc test was used to analyze the difference between independent sets of data and to analyze interaction between 2 factors, that is, ovariectomy effect and AE effect. *p < 0.05, **p < 0.01, ***p < 0.001 compared with SV. ^† p < 0.05, ^†† p < 0.01 compared with OV.

FIGURE 6.

The effect of 50 mg/kg Aloe vera extract supplementation for 12 weeks on cortical bone structure at femora mid‐shaft diaphysis examined by pQCT. Experimental design was similar to that shown in Figure 5. A, bone area; BMC, bone mineral content; BMD, bone mineral density; Ct, cortical; Es.Pm, endosteal perimeter; Ps.Pm, periosteal perimeter; Th, thickness. Results are expressed as means ± SE with individual values. Two‐way ANOVA followed by Tukey's multiple comparisons post hoc test was used to analyze the difference between independent sets of data and to analyze the interaction between 2 factors, that is, ovariectomy effect and AE effect. *p < 0.05, **p < 0.01, *** p < 0.001 compared with SV. ^† p < 0.05 compared with OV.

TABLE 3.

Mechanical property of diaphyseal femur. Rats were divided into non‐treated sham operation (SV) and ovariectomy (OV). SV and OV rats received distilled water (o.p. and i.p., SV and OV control) or Aloe vera extract by oral administration (SAV and OAG) or intraperitoneal injection (SAP and OAP) in the dose of 50 mg/kg, once daily for 12 weeks.

	SV (n = 6)	SAG (n = 6)	SAP (n = 6)	OV (n = 6)	OAG (n = 6)	OAP (n = 5)
Femoral length (mm)	32.90 ± 0.20	33.97 ± 0.11*	33.25 ± 0.21	34.67 ± 0.15*	34.34 ± 0.12*	34.15 ± 0.21*
Maximum load (N)	158.2 ± 5.72	157.7 ± 4.71	162.7 ± 3.19	176.8 ± 5.21	172.1 ± 3.80	163.4 ± 5.65
Yield load (N)	107.9 ± 3.97	105.5 ± 3.59	109.3 ± 3.78	111.4 ± 4.89	107.4 ± 1.93	99.22 ± 3.14
Stiffness (N/mm)	475.9 ± 17.6	494.3 ± 17.6	499.1 ± 10.8	523.2 ± 19.2	500.1 ± 16.1	493.1 ± 13.0
Ultimate displacement (μm)	540.5 ± 16.0	570.8 ± 15.5	539.8 ± 18.6	571.0 ± 28.4	615.9 ± 22.1	579.7 ± 36.9
Yield displacement (μm)	264.1 ± 15.6	267.4 ± 15.5	242.7 ± 11.1	246.7 ± 17.2	266.6 ± 15.4	230.1 ± 9.18
Young's modulus (GPa)	20.98 ± 0.78	21.73 ± 0.86	21.88 ± 0.43	22.99 ± 0.87	22.11 ± 0.64	21.82 ± 0.53

Note: Data was shown as mean ± SE. The difference between groups was analyzed by two‐way ANOVA with Tukey's multiple comparison test.

^* p < 0.05 compared with SV.

Figure 7a showed representative 3D micro‐CT images of L5 lumbar vertebrae. Ovariectomy caused a drastic reduction in trabecular BMD, BMC, bone volume fraction (BV/TV), trabecular thickness (Tb.Th) and an increase in trabecular separation (Tb.Sp) (Figure 7b). AE administration did not have an effect on Tb.BMD; only a tendency to increase in Tb.BMC was observed in the orally gavaged group. AE supplemented via i.p. route OAP was found to increase in Tb.BMC (Figure 7b).

FIGURE 7.

The effect of 50 mg/kg Aloe vera extract supplementation for 12 weeks on lumbar vertebrae 5 (L5) examined by ultra‐high resolution μCT. Experimental design was similar to that shown in Figure 5. (a) representative 3D micro‐CT images of the femur and (b) μCT analysis of trabecular compartment of lumbar vertebrae L5. BMC, bone mineral content; BMD, bone mineral density; BV/TV, bone volume normalized by tissue volume; Tb.Sp, trabecular separation; Tb.Th, trabecular thickness. Results are expressed as means ± SE with individual values. The differences between experimental groups were determined by one‐way ANOVA followed by Tukey post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001 compared with SV. ^† p < 0.05 compared with OV.

4.5. Ovariectomized Rats Had High Osteoclastic Bone Resorption, Which Led to Bone Loss Without Any Change in Bone Formation. AE Supplementation Alleviated Bone Loss by Enhancing Osteoblastic Bone Formation and Suppressing the Hyperactive Osteoclast

Serum levels of bone turnover markers, that is, P1NP (bone formation marker) and TRAP (bone resorption markers) showed that OV rats did not show any change in bone turnover markers. AE supplementation via oral gavage significantly increased P1NP in sham and OV rats (SAG50 and OAG100) (Figure 8a). On the other hand, TRAP in sham and OV rats was not affected by AE (Figure 8b). For this reason, bone microarchitectural and bone cellular changes were further investigated only in orally AE‐treated OAG rats. Representative micrographs of tibiae sections stained with Goldner's trichrome were shown in Figure 8c. Bone histomorphometry analysis confirmed bone loss in OV rats. OV rats exhibited a considerable reduction in BV/TV (12.7% ± 1.05% in Ovx vs. 25.0% ± 0.95% in sham) and trabecular number (Tb.N), and an increase in Tb.Sp. OV‐induced bone loss was due to an increase in osteoclast surface (Oc.S/BS, 2.23 ± 0.22 in OV vs. 0.88 ± 0.09 in sham) and active eroded surface (aES/BS, 1.92 ± 0.3 in OV vs. 0.82 ± 0.07 in sham) (Figure 8d). Although oral 50 mg/kg AE OAG50 had no effect in sham rats, it ameliorated bone loss in OV rats. BV/TV in AE‐treated OV rats (OAG50) was 41% higher than that in non‐treated OV rats (17.9 ± 0.97 in AE‐treated vs. 12.7 ± 0.95 in non‐treated) but was still significantly lower than in AE‐treated sham SAG rats. At the microscopic level, Tb.N in AE‐treated OVx was higher and Tb.Sp was lower than the corresponding values in non‐treated OV rats. The effect of AE was further demonstrated at the cellular level, whereby AE‐treated OV rats exhibited higher Ob.S/BS, lower Oc.S/BS, and lower aES/BS than those of the non‐treated OV group. Bone histomorphometry analysis indicated that 12 weeks of daily 50 mg/kg AE administration could prevent OV‐induced bone loss by enhancing osteoblastic bone formation and suppressing osteoclastic bone resorption. As for two‐way ANOVA analysis, the OV effect and AE supplementation effect each reached statistically significant differences in all bone‐related parameters, except for Tb.Th, and there were interactions between the two factors in BV/TV, Tb.Th, Tb.Sp, Ob.S/BS, and Oc.S/BS (Figure 8d). Overall findings from the present study were summarized in Figure S9.

FIGURE 8.

Oral supplementation of Aloe vera extract alleviated ovariectomy‐induced bone loss by stimulating bone formation and suppressing osteoclastic bone resorption examined by bone histomorphometry. Sham (S) and ovariectomized (O) rats were supplemented with distilled water (SV and OV) or Aloe vera extract (AE). For AE supplemented groups, rats were administered in the dose of 50 mg/kg via either oral gavage (SAG and OAG) or intraperitoneal injection (SAP and OAP). Another group of OV rats was oral administered with 100 mg/kg. Rats were sacrificed 12 weeks post‐supplementation. (a) serum level of bone formation marker, pro‐collagen type 1 and (b) serum level of bone resorption marker, tartrate‐resistant acid phosphatase. (a, b) Results are expressed as means ± SE with individual values. The differences between experimental groups were determined by one‐way ANOVA followed by Tukey post‐hoc test. ^* p < 0.05 compared with SV, ^† p < 0.05 compared with OV, (c) representative images of tibia section stained Goldner's trichrome. Allows indicate trabeculae. Ma, marrow. Scale bar, 1 mm, and (d) bone microstructure and bone cell parameters. Two‐way ANOVA followed by Tukey's multiple comparisons post‐hoc test was used to analyze the difference between independent sets of data and to analyze interaction between 2 factors, i.e., ovariectomy effect and AE effect. ^* p < 0.05, ^** p < 0.01 and ^*** p < 0.001.

5. Discussion

Bone remodeling is a dynamic equilibrium in which bone resorption is tightly coupled with bone formation, maintaining net bone mass [29]. Estrogen has been shown to play an essential role in bone metabolism. The decline in ovarian function during menopause generally leads to bone brittleness, an increased risk of fractures, and metabolic diseases [30, 31]. Most of the anti‐osteoporotic drugs used today, such as bisphosphonates (e.g., alendronate and risedronate) focus on inhibiting osteoclast activity to prevent bone loss. However, the current anti‐osteoporotic drugs induce a number of undesirable effects, for example, esophageal irritation, hypercalcemia, osteonecrosis, and thromboembolic disease [32]. Hence, we aimed to search for alternative forms of medications, such as nutraceutical products that are safe and capable of stimulating osteoblastogenesis while inhibiting osteoclastogenesis in order to prevent bone loss in estrogen deficiency. The present study showed that removal of ovaries from both sides resulted in a marked reduction in 17β‐estradiol and a marked reduction in the weight of uterine horns, bone loss, and gain in body weight compared with sham operation, all of which are consistent with estrogen deficiency.

In addition to osteopathy, a decline in ovarian hormone production is associated with obesity, metabolic syndrome, insulin resistance, and dyslipidemia, all of which are risk factors for type 2 diabetes in both women and female rodents [31, 33]. Furthermore, ectopic fat accumulation in the liver, liver inflammation, and liver oxidative stress, which accelerate the aging process, are observed in postmenopausal women and ovariectomized animals [1, 34]. Consistent with earlier studies [2, 35, 36], we showed that untreated ovariectomized (OV) rats exhibited weight gain as early as the second week post‐surgery, hyperglycemia, impaired glucose tolerance, increased white adipose tissue (i.e., urogenital fat and perirenal fat), dyslipidemia, hepatic fat accumulation with impaired liver function (Figure 1 and Table 1) and these corresponded to an increment of serum insulin level, indicating compromised insulin action in OV rats via inhibition of the IRS1/Akt signaling pathway. Administration of Aloe vera extract (AE) slowed down body weight gain caused by ovariectomy by decreasing fat mass and restoring glucose tolerance to levels observed in the sham group [37]. There were no signs of toxicity when AE was administered either orally or via intraperitoneal injection (Table 1 and Figure S2) indicating that AE is effective and safe when ingested or injected. AE exhibits hypoglycemic and hypolipidemic effects [38, 39]. The hypoglycemic property of AE extract was postulated to result from inhibition of α‐amylase, α‐glucosidase, and dipeptidyl peptidase IV (DPP‐IV) [37]. A previous molecular docking study showed that the active compounds exerting hypoglycemic effects were polyphenolic compounds with sugar moieties (e.g., aloe emodin‐diglucoside, rutin, aloe emodin‐8‐O‐glucoside and acemannan) [37, 40]. Additionally, acemannan and fructans in Aloe vera exhibited prebiotic potentials by modulating gut microbiota. These two polysaccharides increased the population of beneficial bacteria (Bifidobacterium spp.) and promoted the production of acetate, butyrate, and propionate [41]. Bifidobacterium spp. have been found to correlate with improved insulin resistance and obesity [42]. Moreover, we investigated the protein expression of GLP‐1 and GIP in the ileum, the major site of their synthesis, and found that OV rats expressed a higher of GLP‐1 as a result of hyperglycemia with hyperinsulinemia. AE supplementation restored GLP‐1 expression to sham levels and normalized blood glucose levels. As shown in the present study, the higher serum insulin levels in the AE‐supplemented group were probably due to AE ameliorating oxidative stress and preventing the activation of the NLRP3/IL‐1β pathway, previously induced by estrogen deficiency, thereby improving pancreatic islet function and allowing insulin to exert its action on peripheral tissues via activation of the IRS1/Akt pathway (i.e., gastrocnemius and liver) (Figures 1 and 2; Figures S4 and S5). Upon insulin stimulation, the AKT pathway exemplifies a critical mechanism of signal amplification in cellular metabolism. Even a minimal phosphorylation of a subpopulation of AKT proteins can trigger a significant and widespread downstream signaling cascade [43].

It is well established that loss of ovarian hormones increases body weight gain and adiposity in female rats. Although AE supplementation reduced fat accumulation in the liver and urogenital fat depot, it failed to correct the ovariectomy‐induced hypercholesteremia and hypertriglyceridemia (Figure 1 and Tables 1 and 2). It is also known that FGF‐21 is a glucose and lipid regulating hormone that acts on multiple organs such as the liver, heart, skeletal muscle, and pancreas [44]. In humans, circulating FGF‐21 level is elevated in pathological conditions related to metabolic disturbances, such as metabolic syndrome, obesity, dyslipidemia, insulin resistance, and non‐alcoholic fatty liver disease [44, 45]. The elevated circulating FGF‐21 levels in postmenopausal women may result from FGF‐21 resistance, similar to that observed in poor glucose homeostasis caused by pancreatic dysfunction in rodents [46, 47]. The present study showed that AE administration ameliorated metabolic disturbances, decreased fat accumulation, and protected against liver damage. The decrease in circulating FGF‐21 levels in OV rats after AE supplementation indicated a downregulation of FGF‐21 as a compensatory response to the recovery of fat and glucose metabolism. Furthermore, AE administration restored the protein expression of NLRP3 and proinflammatory cytokines, that is, IL‐1β and IL‐6, to normal level in the liver and pancreas and also reduced cell senescence markers in hepatocytes, thus reflecting the recovered organ function (Figure 2). With the same dosage of AE, whether administered by oral gavage or intraperitoneal injection, the anti‐diabetic effects were the same, with only one exception of serum insulin that was slightly higher in the intraperitoneal injected group (Table 2). This could be explained in terms of differences in the bioavailability of the active compound in the bloodstream.

Pharmacokinetic study of aloin showed that it was quickly absorbed (Tmax = 0.25 h) and rapidly cleared from the body (T1/2 = 3.96 h). Meanwhile, in rats given aloin‐containing AE via oral administration, aloin remained in the bloodstream longer (T1/2 increased from 3.96 to 10.16 h), suggesting that other compounds in AE may increase the exposure time of aloin in the plasma through a yet‐unknown mechanism [48, 49]. Additionally, a pharmacokinetic study of aloe‐emodin, an active compound in AE with osteogenic property, showed that aloe‐emodin remained longer in the bloodstream in rats given oral aloe‐emodin compared to intravenous injection [50]. The pharmacokinetics and pharmacodynamics of AE comparing the two routes of administration, that is, oral gavage versus intraperitoneal injection, remain to be fully elucidated.

Although a previous study [38] showed that AE administration improved hypertriglyceridemia and hypercholesterolemia in insulin‐resistant rodents (high fat model and diabetes) with the action being mediated by aloe emodin [51], the present study did not demonstrate this effect in OV rats (Table 1 and Figure S7). However, AE did protect against liver injury, hepatic fat accumulation, and fat deposition in the urogenital fat depot.

Another interesting action of estrogen is related to the intestine. Loss of estrogen increased intestinal permeability and increased the expression of proinflammatory cytokines in immune cells within the intestine under the influence of gut microbiota [52]. Ovariectomy‐induced increase in intestinal permeability was associated with down‐regulation of tight junction proteins, zonula occluden‐1 (ZO‐1) and occludin, and up‐regulation of Claudin‐2 [4], and decreased intestinal calcium absorption [53]. Recently, an in vitro study using the Caco‐2 epithelial cell line showed that AE administration strengthened the intestinal barrier by upregulating the expression of ZO‐1 and suppressing ZO‐1 disruption induced by dextran sodium sulfate [54]. In this study, we found that the fractional calcium absorption, which was compromised in OV rats, was fully restored by AE supplementation (Figure 3). We postulated that the increase in intestinal calcium absorption was due to three possible mechanisms, that is, (i) the prebiotic effect of AE, which indirectly improved gut microbiota by promoting the growth of short‐chain fatty acid (SCFA)‐producing bacteria such as Bifidobacterium and Lactobacillus spp. [23, 41], (ii) acemannan, a polysaccharide in Aloe vera , was broken down into SCFA (acetate, propionate and butyrate) [41, 55], which promoted intestinal calcium absorption [24], and (iii) an increase in the circulating level of 1,25(OH)₂D₃, which stimulates calcium absorption. AE's influence on 1,25(OH)₂D₃ production may have been mediated indirectly through its antioxidant and anti‐inflammatory effects on the kidney. Estrogen deficiency leads to the accumulation of reactive oxygen species (ROS) in several organs including the kidneys, causing oxidative stress and low‐grade chronic inflammation, all of which could be reversed by AE supplementation, and therefore, restoring kidney function and synthesis and secretion of 1,25(OH)₂D₃ (Table 2).

Several in vitro studies have suggested that bioactive compounds from Aloe vera have potential benefits on bone health. Aloe‐emodin has been shown to induce chondrogenic differentiation in mouse chondrogenic ATDC5 cells through BMP‐2 and MAPK‐signaling pathways [56]. Moreover, acemannan‐treated Sprague–Dawley rats in a tooth extraction model exhibited higher bone mineral density and faster bone healing in vivo [9], suggesting that acemannan could be responsible for the AE's stimulatory effect on bone formation. The present results were consistent with previous reports [3, 57, 58] that OV rodents, without any change in circulating bone turnover markers, exhibited bone loss primarily in the trabecular region and a slight decrease in cortical shell of the proximal femur compared to age‐matched sham controls. They also displayed higher femoral length and diaphyseal mid‐shaft cross‐sectional bone area without changes in mechanical properties. Fonseca and co‐workers (2012) and Flieger and co‐workers (1998) explained that bone strength was mostly linked to bone size, diaphyseal mid‐shaft cross‐sectional bone area, and femoral neck geometry. Despite the decrease in bone volume and mineralization following estrogen loss, bone strength was not reduced. Hence, the increase in femur size and change in femoral neck geometry in OV rats likely compensated for bone loss to some extent, thereby preserving bone strength [57, 59]. Additionally, the increases in diaphyseal cortical bone mass, bone area and cortical thickness were found to be associated with increased in body weight [60]. In agreement with other finding in OV rats [61], AE administration partially prevented OV‐induced bone loss as shown by significant increase in trabecular BMD and BMC (Figures 4 and 5). The decrease in cortical BMD and BMC in AE supplemented OAP group was probably due to lower body weight, which, in turn, reduced the obesity‐induced diaphyseal bone adaptation. The greater trabecular bone density in the OAP group has potential clinical significance such as treatment of trabecular osteopenia and prevention of fractures at trabecular sites. AE administration prevented the deterioration of trabecular microarchitecture in the metaphyseal tibia by enhancing osteoblastic bone formation and inhibiting osteoclastic bone resorption. According to the previous in vitro and in vivo studies [61], Aloe vera polysaccharides directly stimulated osteoblast differentiation from multidirectional‐differentiated adipose‐derived stromal cells through BMP2/Smads signaling pathway. It stimulated osteoblast function (evidenced by increased alkaline phosphatase activity, alizarin red staining and the expression of collagen 1A1 and osteopontin), while inhibiting adipogenesis by downregulating the expression of PPAR𝛾. Another in vitro study by Pengjam and co‐workers (2016) [8] showed that phenolic compound, Aloin, in AE prevent osteoclastogenesis in RAW264.7 cell line. These dual actions of AE on bone were quite different from most anti‐osteoporotic agents which mainly exhibit only the antiresorptive action [62] (Figures 4, 5, 6, 7, 8).

The limitations of the study were (i) the active ingredients in AE were not identified or quantified, (ii) the pharmacodynamics and pharmacokinetics of the active ingredients were not provided, (iii) this study did not include active ingredients as an explicit control, (iv) estrogen deficiency by ovariectomy could not fully recapitulate the post‐menopausal condition, (v) there was a lack of information regarding the long‐term outcomes of AE supplementation on bone and metabolic disturbances as well as the long‐term side effect of AE.

In conclusion, the present in vivo study demonstrated the anti‐diabetic and osteogenic properties of Aloe vera extract. When supplemented at the doses of 50 or 100 mg/kg AE by oral gavage or intraperitoneal injection for 8 weeks, AE slowed down the body weight gain, decreased urogenital fat depot and fat accumulation in the liver, restored glucose tolerance and prevented the OV‐induced bone loss. To the best of our knowledge, this was the first reported of the osteoblastogenic and anti‐osteoclastogenic effects of Aloe vera in osteoporosis animal model. Lastly, the study provided a clear evidence supporting the postulation that Aloe vera has potential to be used as a therapeutic intervention to reduce the need for drugs in the treatment of diabetes and osteoporosis in postmenopausal women.

Author Contributions

Panan Suntornsaratoon: conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, supervision, validation, visualization, writing – original draft, writing – review and editing. Wajathip Bulanawichit: investigation. Wikanda Chimlek: investigation. Wanwipa Saeten: investigation. Waraporn Sorndech: investigation. Wiriyaporn Sumsakul: investigation. Suchinda Malaivijitnond: resources, writing – review and editing. Vitoon Saengsirisuwan: resources, writing – review and editing. Nateetip Krishnamra: formal analysis, resources, writing – review and editing. Narattaphol Charoenphandhu: conceptualization, formal analysis, funding acquisition, investigation, methodology, resources, supervision, validation, visualization, writing – review and editing.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Data S1.

Data S2.

Data S3.

Suntornsaratoon P., Bulanawichit W., Chimlek W., et al., “Carbohydrate‐Rich Fraction of Aloe vera (L.) Burm.f. Extract Mitigates Bone Loss and Improves Metabolic Disturbance in Estrogen‐Deficient Rats,” Pharmacology Research & Perspectives 13, no. 4 (2025): e70148, 10.1002/prp2.70148.

Data Availability Statement

The datasets generated and/or analyzed during the current study are not publicly available due to future patent filing, but are available from the corresponding author on reasonable request.