Evaluation of the combined effects of Morinda citrifolia fruit and Averrhoa bilimbi leaves on hair growth in rabbits

Abstract

This study was conducted to evaluate the impact of a combination of Morinda citrifolia fruit extract (MCFE) and Averrhoa bilimbi leaf extract (ABLE) on hair growth in rabbits. The study included seven treatment groups: (1) control group (distilled water), (2) positive control (minoxidil 5%), (3) treatment 1 (MCFE 10%), (4) treatment 2 (ABLE 10%), (5) treatment 3 (MCFE 5% + ABLE 5%), (6) treatment 4 (MCFE 7.5% + ABLE 2.5%), and (7) treatment 5 (MCFE 2.5% + ABLE 7.5%). Hair length measurements were taken on days 7, 14, 21, and 28, and the average daily growth rate was calculated by dividing the change in hair length by the number of days. Hair weight was measured on day 28. Data analysis was conducted using both two-way and one-way ANOVA, and statistical significance was determined at P < 0.05. The treatment group 4 (MCFE 7.5% + ABLE 2.5%) showed a significant difference in hair growth compared to the normal control, positive control, and other treatment groups (P < 0.05). The group exhibited a 19.69 ± 0.38 mm increase in hair length, with a daily growth rate of 0.88 mm/day, and an increase in hair weight of 0.33 ± 0.003 g. The combination of MCFE and ABLE demonstrated potential as an anti-hair loss agent, effectively enhancing both hair length and weight.

INTRODUCTION

Hair loss, often resulting in baldness, is characterized by the absence of hair in certain areas or the limited regrowth of hair.[1] Several factors such as genetics, hormonal imbalances, stress, health conditions, inadequate nutrition, and medications can play a role in the development of hair loss.[2] Minoxidil and finasteride are standard treatments for hair loss.[3] However, both can have negative effects in the long term, such as adverse skin reactions, headaches, vertigo, weakness, and edema.[4]

Currently, treatment with natural ingredients is increasingly preferred over synthetics due to their safety and reduced adverse effects.[5] Native Indonesian plants, including Morinda citrifolia fruit and Averrhoa bilimbi leaves, contain chemical compounds that impact hair growth. The alkaloids found in M. citrifolia, such as piperazine and piperidine, can prevent alopecia by increasing hair length and weight.[6] Research by Priatna et al.[7] revealed that the flavonoids in A. bilimbi leaves can stimulate hair growth and lengthening in rabbits. The combination of M. citrifolia and A. bilimbi can provide benefits in increasing the therapeutic effect. These two herbs work together to complement each other and promote hair growth.

To date, no scientific studies have explored the potential anti-hair loss effects of a combination of M. citrifolia fruit and A. bilimbi leaves. This research aims to evaluate the effectiveness of this combination in promoting hair growth, with a focus on hair length, growth rate, and weight.

MATERIALS AND METHODS

Plants and chemical materials

The standardized extract of Morinda citrifolia fruit extract (MCFE) (SLACG1001) was acquired from PT. Indo Plant Semarang, Indonesia. A. bilimbi leaves were sourced from Lembang district, Bandung, Indonesia, and authenticated with number 028/KET/LBF/G/2021 by the Biology Pharmacy Laboratory at Universitas Muhammadiyah Kuningan, Indonesia. Methanol, hydrochloric acid, sulfuric acid, ferric chloride, and all chemical reagents were provided by Sigma-Aldrich, Germany.

Animals

In this study, male rabbits aged 2–4 months (1.5–2 kg BW) were used. We calculated the number of rabbits using the Federer formula,[8] as follows:

(t − 1) (n − 1) ≥ 15

t: Treatment groups

n: Number of repetitions of each treatment.

The rabbits underwent a 14-day acclimatization period in the laboratory to adjust to the environment and were kept under standard conditions. All animal-related procedures were approved by the Research Ethics Committee of Bakti Tunas Husada University, West Java, Indonesia (Approval No. 057/E.02/KEPK-BTH/VI/2023).

Extract preparations

The A. bilimbi leaves were dried and powdered. One kilogram of dry A. bilimbi leaf powder was extracted using 10 L of 70% ethanol for 24 h through maceration. The filtrate was mixed and evaporated twice after remaceration to obtain a thick extract.[7] The extract was stored at 4°C for use in this study.

Physicochemical and heavy metal analysis

The physicochemical analysis involved measuring parameters such as moisture content, total ash, acid-insoluble ash, ethanol-soluble ash, and water-soluble ash content. For heavy metal analysis, atomic absorption spectroscopy (DW-AA320N, Drawell) was employed, using argon as a carrier gas with a flow rate of 1 ml/min. The concentration of primary heavy metals was determined using 500 mg of the extract.[9]

Qualitative phytochemical screening

A qualitative phytochemical test was performed on MCFE and A. bilimbi leaf extract (ABLE) to identify various phytoconstituents.[10,11]

Test of hair growth activity

The test in this study refers to the methods of Tanaka et al.[12] with modification. In this test, the rabbit’s back was shaved and divided into several sections, namely: P1: normal control (distilled water), P2: positive control (minoxidil 5%), P3: treatment 1 (MCFE 10%), P4: treatment 2 (ABLE 10%), P5: treatment 3 (MCFE 5% + ABLE 5%), P6: treatment 4 (MCFE 7.5% + ABLE 2.5%), and P7: treatment 5 (MCFE 2.5% + ABLE 7.5%). One milliliter of extract was applied twice daily for 28 days. Hair length was measured on days 7, 14, 21, and 28,[13] and the average growth daily (AGD) rate was obtained using the formula: the average hair length divided by days. After 28 days, all hair in the test area was shaved, and hair weight was measured.[14] Figure 1 shows the location of the treatment area on the rabbits. The comparison of concentrations in this study is based on previous research, where MCFE and ABLE showed optimal hair growth effects at a concentration of 10%.[7,15]

Figure 1.

The position of treatment area hair growth activity of MCFE and Averrhoa bilimbi leaf extract combination on the rabbit. P1 (normal control), P2 (positive control), P3 (treatment 1), P4 (treatment 2), P5 (treatment 3), P6 (treatment 4), and P7 (treatment 5)

Statistical analysis

Data were expressed as mean ± standard deviation. For the analysis of hair length differences between the treatment groups, two-way ANOVA was applied, while one-way ANOVA was used for hair weight comparison. Tukey’s multiple comparison test was then conducted using GraphPad Prism 9.5.1 software (GraphPad Software, San Diego, USA). In cases where data were not normally distributed or homogeneous, the Kruskal–Wallis test followed by the Mann–Whitney test was used. Statistical significance was defined at P < 0.05.

RESULTS

Analysis of physicochemical and heavy metal

The physicochemical evaluation of MCFE revealed the following composition: moisture content – 4.90%, total ash – 10.15%, acid-insoluble ash – 2.85%, ethanol-soluble ash – 67.83%, and water-soluble ash – 96.55% (w/w). These results were consistent with the standards set by Materia Medica and the Indonesian Herbal Pharmacopeia.[16,17]

The physicochemical evaluation of ABLE revealed the following composition: moisture content – 4.27%, total ash – 0.02%, acid-insoluble ash – 23.80%, ethanol-soluble ash – 52.89%, and water-soluble ash – 99.63% (w/w) [Table 1]. These results align with the standards established by Materia Medica.[17]

Table 1.

Physicochemical analysis of Morinda citrifolia fruit extract and Averrhoa bilimbi leaf extract

Parameters		Results (% w/w)
		MCFE		ABLE
Moisture content		4.90		4.27
Total ash content		10.15		0.02
Acid-insoluble ash content		2.85		23.80
Ethanol-soluble ash content		67.83		52.89
Water-soluble ash content		98.55		99.63

MCFE: Morinda citrifolia fruit extract, ABLE: Averrhoa bilimbi leaf extract

Meanwhile, the concentration of heavy metals such as arsenic, mercury, lead, and cadmium is >0.001 µg/g [Table 2]. Heavy metal testing was conducted to evaluate the safety and quality of the extract. This analysis helps identify harmful substances such as arsenic, mercury, lead, and cadmium.[18,19] The results are consistent with the safety standards outlined by the WHO.[20,21]

Table 2.

Heavy metal analysis

Heavy metals		Result (μg/g)
Arsenic		<0.001
Mercury		<0.001
Lead		<0.001
Cadmium		<0.001

Phytochemical screening

The qualitative phytochemical screening results indicate that MCFE and ABLE contain phenols, triterpenoids, alkaloids, tannins, saponins, and flavonoids [Table 3].

Table 3.

Morinda citrifolia fruit extract and Averrhoa bilimbi leaf extract qualitative phytochemical analysis

Metabolite		Results
		MCFE		ABLE
Phenols		+		+
Triterpenoids		+		+
Alkaloids		+		+
Tannins		+		+
Saponins		+		+
Flavonoids		+		+

MCFE: Morinda citrifolia fruit extract, ABLE: Averrhoa bilimbi leaf extract

Hair growth activity of MCFE and Averrhoa bilimbi leaf extract combination

All groups exhibited hair growth on days 7, 14, 21, and 28, with significant differences observed between the positive control and treatment groups. The treatment 4 group (MCFE 7.5% + ABLE 2.5%) exhibited the most significant increase in hair length, measuring 19.69 ± 0.47 mm, in comparison to the positive control group, which showed 11.13 ± 0.85 mm (P < 0.0001) [Table 4]. Similarly, hair weight analysis for the MCFE and ABLE combination demonstrated significant increases in all the treatment groups. The treatment 4 group exhibited a hair weight of 0.3300 ± 0.0033 g, which was significantly higher than the normal control (0.1083 ± 0.0190 g) and the positive control (0.1875 ± 0.0040 g) (P < 0.0001).

Table 4.

Hair growth activity of the Morinda citrifolia fruit extract and Averrhoa bilimbi leaf extract combination

Groups		Hair length (mm)±SD								Hair weight (g)±SD - Day 28
		Day 7		Day 14		Day 21		Day 28
Normal control (distilled water)		3.15±0.11		5.01±0.07		5.57±0.34		7.95±0.31		0.1083±0.0190
Positive control (minoxidil 5%)		6.02±0.76*		7.52±0.46*		9.31±0.42*		11.13±0.85*		0.1875±0.0040*
Treatment 1 (MCFE 10%)		7.17±0.43*,a		7.89±0.43*,a		11.15±0.43*,a		12.47±0.47*,a		0.2084±0.0180*,a
Treatment 2 (ABLE 10%)		7.74±0.52*,a		8.30±0.57*,a		10.86±0.32*,a		12.81±0.68*,a		0.2299±0.0329*,a
Treatment 3 (MCFE 5% + ABLE 5%)		7.81±0.60*,a		9.80±0.79*,a		12.69±0.74*,a		14.01±0.83*,a		0.2346±0.0033*,a
Treatment 4 (MCFE 7.5% + 2.5%)		8.22±0.93*,a		11.22±0.93*,a		18.00±0.73*,a		19.69±0.38*,a		0.3300±0.0033*,a
Treatment 5 (MCFE 2.5% + ABLE 7.5%)		7.88±0.26*,a		10.90±0.28*,a		17.70±0.45*,a		18.67±0.65*,a		0.3032±0.0097*,a

*P<0.05 normal control versus treated groups, ^aP<0.05 positive control versus treated groups, values are presented as mean±SD. SD: Standard deviation, MCFE: Morinda citrifolia fruit extract, ABLE: Averrhoa bilimbi leaf extract

The AGD rates for hair growth also differed significantly across groups. The treatment 4 group achieved a higher AGD of 0.88 mm/day compared to the normal control group (0.33 mm/day) and the positive control group (0.55 mm/day) (P < 0.0001). Figure 2 shows the average daily hair length growth.

Figure 2.

Growth rate parameters based on average growth daily calculation. Each value represents the mean ± standard deviation of four repetitions. P < 0.05 (significance). *in comparison to normal control, ^ain comparison to positive control

DISCUSSION

Findings from this research suggest that the combination of MCFE and ABLE effectively promotes hair growth in rabbits. Rabbits and humans share similar hair growth cycles, but their durations and biological mechanisms involved can differ significantly. In addition, their convenient body size makes testing easier and less stressful.[22]

In this study, we used minoxidil 5% for the control-positive group. Minoxidil is a standard treatment for hair loss and alopecia, as it prolongs the hair growth phase, increases follicle cell proliferation, and stimulates dermal papilla cells.[23] Previous research has shown that minoxidil treatment (2%–5%) effectively alters the anagen–telogen ratio and enhances hair growth.[24]

The phytochemical compounds in MCFE and ABLE align with previous studies, indicating the presence of flavonoids, alkaloids, terpenoids, phenols, tannins, and saponins.[7,25] Phenolic protects hair follicles from damage caused by oxidative stress and reduces the duration of the telogen phase.[26,27] Flavonoids increase the anagen phase and regulate the transition from anagen to catagen by increasing blood circulation to the hair follicles.[28] Alkaloids modulate the catagen transition, reduce the duration of the telogen phase, and inhibit dihydrotestosterone activity, while tannins strengthen hair follicles by prolonging the anagen phase.[29]

The treatment 4 group in this study exhibited a better effect compared to the other combination treatment groups, possibly due to MCFE having a similar mechanism to minoxidil. Studies suggest that MCFE can prevent hair loss by enhancing hair follicle activity, promoting the transition from the telogen phase to the anagen phase, and nourishing hair follicles.[30] Antioxidants in MCFE may protect hair follicles from oxidative stress.[31] In addition, the higher concentration of MCFE in this group led to a more significant effect compared to the other groups. The results of this study highlight the anti-hair loss potential of the combination of MCFE and ABLE by increasing hair length and weight. However, it is worth noting that no histological analysis of hair dermal papilla cells was conducted in this study.

CONCLUSION

The combination of MCFE and ABLE may have potential hair growth effects by accelerating hair growth, increasing the hair growth rate, and increasing hair weight. Therefore, this combination can be developed into an herbal preparation to treat hair loss.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

This article is supported by the Ministry of Education, Culture, Research, and Technology through the PDP Grant 2024.