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. 2023 Jun 8:1–16. Online ahead of print. doi: 10.1007/s11101-023-09880-1

Chimaphila umbellata; a biotechnological perspective on the coming-of-age prince’s pine

Urooj Ali 1,2, Muhammad Mustajab Khan 2, Naveera Khan 2, Rida tul Haya 2, Muhammad Usama Asghar 2, Bilal Haider Abbasi 2,3,✉
PMCID: PMC10249550  PMID: 37359710

Abstract

Chimaphila umbellata has been studied for almost two centuries now, with the first paper exploring the phytochemistry of the plant published in 1860. Almost all contemporary studies focus on the biotechnological advances of C. umbellata including its utilization as a natural alternative in the cosmetic, food, biofuel, and healthcare industry, with a special focus on its therapeutic uses. This literature review critically investigates the significance and applications of secondary metabolites extracted from the plant and presses on the biotechnological approaches to improve its utilization. C. umbellata is home to many industrially and medicinally important phytochemicals, the majority of which belong to phenolics, sterols, and triterpenoids. Other important compounds include 5-hydroxymethylfurfural, isohomoarbutin, and methyl salicylate (the only essential oil of the plant). Chimaphilin is the characteristic phytochemical of the plant. This review focuses on the phytochemistry of C. umbellata and digs into their chemical structures and attributes. It further discusses the challenges of working with C. umbellata including its alarming conservation status, problems with in-vitro cultivation, and research and development issues. This review concludes with recommendations based on biotechnology, bioinformatics, and their crucial interface.

Keywords: Chimphila umbellate, 15-all-cure herb, Chimaphilin, Methyl salicylate, Plant biochemistry, In-vitro cultivation

Introduction

Chimaphila umbellata (shown in Fig. 1) has been studied for almost two centuries now, with the first paper exploring the phytochemistry of the plant published in 1860. Almost all the contemporary studies focus on the biotechnological advances of C. umbellata including its utilization as a natural alternative in the cosmetic, food, biofuel, and healthcare industry, with a special focus on its therapeutic applications. Peacock (1892) evaluated the crystalline compound that was extracted from C. umbellata by distilling it with water. The compound Chimaphilin gave the genus its name. The investigation also revealed that the quantity of Chimaphilin, already present in trace amounts in the plant, decreases from roots to fruits, with the latter not producing any of it (Peacock 1892). C. umbellata, a Eurasian plant, is found in almost all of North America and is distributed throughout the cool temperate northern hemisphere.

Fig. 1.

Fig. 1

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The plant; Chimaphila umbelleta; a the flowering plant; b single flower; c flowers; d flower buds; e young fruits (capsules).

Source: Flora of Quebec (2023)

It belongs to the Ericaceae family and is commonly known as Pipsissewa, prince’s pine, or umbellate wintergreen. It is a shrub and grows up to 35 cm with whorled or opposite leaves (Das et al 2022). It is an evergreen plant with an umbel or spring of flowers in the latter parts of summer and fall. It is a fully hardy plant that can withstand harsh temperatures like − 15 °C. It grows in moist soil and requires full shade for primary growth and development, and a semi-shade (a photoperiod) for the growth and development of its evergreen leaves (Freeman 2022). This partitioned growth requirement of C. umbellata has also been demonstrated by Figura et al. (2018) in their study of in-vitro axenic germination of mixotrophic Pyroloideae and their ontogenetic development.

The plant bears stolons (leading to the development of horizontal stems that spread over the ground and produces vertical stems and adventitious roots at the nodes), which along with its dwarf spreading quality make it a decent ground cover (Freeman 2022). Along with this significant horticulture use of C. umbellata, it is also utilized in perfumery due to its delicate smell produced by methyl salicylate, the essential oil of the plant (Clark 1999). The importance of methyl salicylate is further discussed, however, this review, for the most part, discusses the phytochemicals of C. umbellata and their therapeutic potential. With a medicinal rating of 3/5 and an edible rating of 2/5 (Plants for a future 2022), Pipsissewa has been known to treat urinary tract infections, fluid retention problems, bladder stones, epilepsy, anxiety, cancer, spasms, and skin sores and blisters for hundreds of years (Chadde 2020).

The leaves of Pipsissewa are used in condiments, candy extracts, flavoring of beer, and tea. Apart from these, the leaves or their extracts are utilized to treat skin sores and boils. Furthermore, its stems and roots are used in homeopathic decoctions utilized in the treatment of the problems discussed above. The therapeutic potential of C. umbellata is due to its wide array of phytochemical production including arbutin, sitosterol, ursolic acid, glycoside, hydroquinone, and essential oil (Plants for a future 2022). Recent studies have focused on this potential and have examined boils of C. umbellata against tuberculosis (Pathak et al. 2021), the plant’s antiproliferative effect against MCF-7 cancer cells (Das et al. 2022), and its activity against cardiovascular diseases.

Moreover, C. umbellata has been included in the 15-herb cure-all bitters, a book on 15 herbs that are said to treat all diseases (Parsons 2011). The leaves of C. umbellata when used externally, are rubefacient, and work against cardiac problems, scrofula, kidney issues, and chronic rheumatism. Nonetheless, its long-term utilization as well as excessive quantity is reportedly linked with certain side effects like ringing in the ears, confusion, vomiting, and seizures (Kusheev et al. 2020). Currently, only the leaves of the plant are said to be officinal, but work is being done on the utilization of the whole plant against various diseases. The homeopathic use of C. umbellata requires it in the liquid form, as mother tincture, and is commercialized by many countries’ homeopathic experts. The plant in its liquid form is said to rapidly assimilate in the body with the whole process taking 1–4 min, whereby 85–90% of the extract is assimilated in the first 25 s. The body directly absorbs 98% of the medicinal components of the tincture, and the liquid form satisfies the user’s satiety because of its natural aroma (Freeman 2022).

In-vitro cultivation of C. umbellata

Not much work has been done on Pipsissewa’s in-vitro cultivation owing to many challenges that will be addressed further in this section. However, Figura et al. (2018) have worked on its cultivation along with other plants of the family. Ripe capsules from wild fruits were retrieved and the seeds were collected. The authors tested nine media for the cultivation of seedlings and noted that only Knudson C medium (with activated charcoal), pH adjusted to 5.8 using NaOH, gave positive results and a few seedlings of C. umbellata were germinated. No seedling germinated on any of the other mediums. 1000 mg/L activated charcoal was added to the Knudson medium to improve the growth and germination of C. umbellata. Since the plant is a mycorrhizal symbiont, complex sucrose was substituted by 100 mM glucose to mimic the carbon provided by the fungi in natural conditions. Also, since the plant is a slow-growing herb, 0.01/0.1/1 mg GA3 (gibberellic acid 3) was added to the media to test its effect on the plant elongation (Figura et al. 2018).

Seed disinfection upon germination was carried out by treating the plants with 70% ethanol for five minutes, then washing them 3 times with deionized water, followed by treatment with 2% sulfuric acid and calcium oxychloride for 10 min each, and washing them thrice with deionized water. The germination rate of the seedlings reportedly increased 4 times after treatment with sulfuric acid for 10 min (Figura et al. 2018). As discussed above, the primary development requires dark conditions, so the seedlings were incubated in dark at 4 ºC. When growing shoots were noted via microscopy, they were transferred to light with a photoperiod of 16 h light/8 h dark to produce leafy shoots. The regular potting process with coarse expanded perlite, fine pine bark, loamy soil, and pumice gravel (1:2:1:1) was performed and the pots were kept on a windowsill at room temperature packed in polyethylene to maintain air humidity (Figura et al. 2018).

Upon cultivation, the extraction of phytochemicals was performed. To determine which extraction method will be used, a series of factors are considered. These include stability to heat, nature of the solvent, cost of the drug, extraction duration, the final volume required, and the intended use (Belwal et al. 2018). Based on these factors, around 11 menstrua are utilized in various extraction methods with n-hexane being the least polar and water being the most polar.

During the process of extraction, if a researcher wants to choose five solvents, it is a common practice to select two solvents that have low polarity levels (n-hexane, chloroform), two that have medium polarity levels (dichloromethane, n-butanol), and one with the highest polarity level (water). The first extract of Chimaphila umbellata was produced using petroleum ether extract, stronger ether extract, absolute alcohol extract, water extract, alkaline (NaOH) water extract, acidulated (HCl) water extract, Starch, Moisture, and Ash (Peacock 1892). To further decide on the use of solvents, factors including selectivity, safety, reactivity, cost, recovery, viscosity, and boiling temperature are kept in view (Efthymiopoulos et al. 2018). Based on the extraction of phytochemicals, Fig. 2 provides the characteristic groups of these extracts, adapted from the work of Erb & Kliebenstein (2020).

Fig. 2.

Fig. 2

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Phytochemical categories extracted from plants

Phytochemicals extracted from C. umbellata

Phenolic compounds

The compounds shown in Fig. 3 have been extracted from C. umbellata by Yu et al. (2021a, 2021b). Phenolic compounds have an aromatic ring with one or more hydroxyl groups and an R group. These compounds are produced by Pentose phosphate, shikimate, and phenylpropanoid pathways and account for 40% of the organic carbon of the biosphere (Cosme et al. 2020). Phenolics are found in vascular plants, bryophytes, algae, fungi, and bacteria. They form strong hydrogen bonds, are soluble in water with high boiling points, exist as colorless liquids or white solids, and are highly toxic (Rocchetti et al. 2022). Phenolics are known for broad therapeutic potentials including anti-oxidant activity that adsorb or neutralize free radicals due to their redox properties; an anti-cancerous activity that enhances the immune system to recognize and destroy cancer cells; anti-bacterial activity that inhibits biofilm production and promotes beta-lactamase inhibition of microbes; an anti-UV potential due to pigments natural to phenolics (Cosme et al. 2020), which absorb UV and protect the human skin; an anti-inflammatory activity promoting radical activities and modulation of enzymes; and cardioprotective activities leading to a reduction of hyperlipidemia, inhibition of elevated BP, and so on.

Fig. 3.

Fig. 3

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Phytochemicals extracted from Chimaphila umbellata. a Quercetin, b Kaempferol, c Daucosterol, d Alpha Amvrin, e Beta sitosterol, f Ursolic acid, g 5-Hydroxymethylfurfural, h Methyl salicylate, i Isohomoarbutin, j Chimaphilin

Quercetin

Flavonoids are an important type of hydroxylated phenolic compounds that contain an aromatic ring structure. Quercetin is a flavanol, which is a subtype of flavonoid. It contains five hydroxyl groups that contribute to the compound’s biological activities and derivatives. It is a bioactive natural compound (D'Andrea 2015). Quercetin is yellow-colored, a crystalline insoluble substance that has a bitter taste. It is slightly soluble in aqueous alkaline solutions, glacial acetic acid, and alcohol, despite its insolubility.

Quercetin aids several physiological processes in plants, like pollen growth, photosynthesis, antioxidant machinery, and seed germination. It also gives rise to proper plant development and growth. Against several biotic and abiotic stresses, quercetin provides tolerance to plants. The biosynthesis of quercetin is carried out by the phenylpropanoid metabolic pathway. (Singh et al. 2021). Nowadays quercetin is widely used as a nutritional supplement and for several diseases such as obesity/diabetes and circulatory dysfunction as well as mood disorders as a phytochemical remedy. The antioxidant, anti-inflammatory, anticancer, antidiabetic, antimicrobial, wound healing, and cardiovascular effects of quercetin have been investigated extensively.

Recently, the anticancer activity of quercetin against various cancer cell lines has been reported. Interleukin 6 (IL-6) and C-reactive protein (CRP) are common inflammatory markers, and uplifted levels of IL-6 and CRP are interconnected with elevated risk for depression and mental distress (Wium-Andersen et al. 2013; Khandaker et al. 2014). Animal studies have shown that IL-6 expression levels could be significantly elevated through the administration of lipopolysaccharide both in the spleen and the brain, resulting in depressive-like behavior. It was recently shown that quercetin might protect from stress-induced anxiety and depression-like behavior in male mice, in addition to improving memory in these animals. (Samad et al. 2018). Quercetin can decrease the level of stress-induced brain corticotrophin-releasing factor (CRF) which is associated with depression and anxiety.

Kaempferol

Kaempferol is a Tetrahydroxyflavone, which contains four hydroxy groups that are present at positions 3, 5, 7, and 4. In plants, kaempferol acts as a precursor molecule for the biosynthesis of viral respiratory cofactor ubiquinone (Soubeyrand et al. 2018). Kaempferol can reduce the risk of chronic diseases, particularly cancer. kaempferol impedes cancer cell growth and angiogenesis and instigates apoptosis of cancer cells, but contradictorily, kaempferol appears to conserve normal cell viability, in some cases exerting a protective effect (Silva dos Santos et al. 2021).

Kaempferol administration increased bone density in ovariectomized osteoporosis rats, promoted osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), increased RUNX2, Osterix, and CXCL12 expression, and decreased miR-10a-3p expression (Sekaran et al. 2022; Soubeyrand et al. 2018). It is confirmed that miR-10a-3p and CXCL12 bind to each other in the possible mechanism analysis, and that Kaempferol promoted BMSC osteogenic differentiation and alleviated osteoporosis via lowering miR-10a-3p and increasing CXCL12 (Gan et al. 2022). By lowering miR-10a-3p and increasing CXCL12, Kaempferol promoted BMSC osteogenic differentiation and alleviated osteoporosis, according to findings (Gan et al. 2022; Sekaran et al. 2022; Wu et al. 2021).

Sterols

Sterol is the plants’ cholesterol. Their high amounts are present in seeds, nuts, and vegetable oils. These are normally used to lower the cholesterol level in the body. Sterol is used to cure various diseases like cancers and diabetes etc. (Gylling et al. 2014). along with many positive systematic effects. These plant cholesterols fall into two categories: sterols and stanols. Both sterols and stanols molecules have four rings along with a hydroxyl group on the first ring and an alkyl radical R group on the last ring (Gylling et al. 2014). The major difference between sterols and stanols is the presence of a double bond in the second ring. In Chimaphila umbellata the extract contains two vital sterols named Beta-sitosterol and daucosterol.

Beta-sitosterol

BS is a phytosterol that follows the mevalonate pathway. It is a white, waxy powder with a characteristic odor. It is hydrophobic in nature and soluble in organic solvents like alcohol. Its molecular mass is 414.18kj/mole and its chemical formula is C29H50O (Gupta 2020). It is used as a food additive and has great therapeutic potential along with a lot of systematic effects. BS was known as an orphan phytosterol until 2017 due to a lack of research work on it. Due to continuous ignorance from WHO, FAO and EFSA it is termed as an “ORPHAN BS”. Beta-sitosterol proved its importance by showing that it is natural, safe, and effective and has potential benefits. It is termed GRAS (Generally Regarded as Safe). In the experiments on rats, it did not cause toxicity (Gupta 2020).

BS has antioxidant properties (Choi et al. 2003; Paniagua-Pérez et al. 2005). It is also shown that it modulates antioxidant enzymes and estrogen receptors (Song et al. 2000). It reduces the free radicles and hydrogen peroxide. It also decreases the activities of glutathione peroxidase and Mn sulphoxide mutase (Vivancos & Moreno 2005). It has a major role in blood vessel formation, so it is helpful in wound healing. There is evidence that blood vessel creation occurs during ischemia; however, more research about the viability of using BS as a treatment agent for ischemic stroke has not been carried out (Choi et al. 2003).

BS is used to prevent cardiovascular diseases (Retelny et al. 2008). FDA has approved it for hyperlipidemia (Yeshurun & Gotto 1976). It decreases the cholesterol level in the plasma by displacing it from micelles (Plat & Mensink 2005), (Sugano et al. 1977; Plat et al. 2005; Ellegård et al. 2008). To increase the potency of other statins it is added in combination with other statins (Richter et al. 1996). For example, the combination of BS with simvastatin increases its potency (Rosenblat et al. 2013).

BS performs anti-inflammatory activity in aortic cells (Loizou et al. 2010). It reduces the secretions of pro-inflammatory cytokines, TNF-α as well as edema (Gupta et al. 1980; Bouic et al. 1996); Bouic & Lamprecht 1999). On the other hand, it also increases the anti-inflammatory cytokines (Valerio & Awad 2011). According to the findings of Liz et al. (2013), BS either inhibits or does not inhibit levels of IL-1β and TNF-α by rising calcium absorption in activated neutrophils in a concentration- and time-dependent manner via L-type voltage-dependent calcium channels, intracellular calcium, phosphoinositide kinase-3, and microtubule modulation. This boosts the anti-inflammatory function. Apart from these, BS is reported to have anti-arthritic, anti-pyretic, anti-cancerous, anti-pulmonary, anti-diabetic, anti-microbial, and anti-HIV activities (Liz et al. 2013).

Daucosterol

Daucosterol is a phytosterol and it is the extension of sitosterol to which a beta-D-glucopyranosyl residue is attached at the 3’ position by a glycosidic linkage. In short, it is termed the glucoside of beta-sitosterol or simply sitoglucide. Its molar mass is 576.859 g/mole with a molecular formula as; C35H6006 (Zeng et al. 2017). It works like beta-sitosterol in a therapeutic sense. It shares its therapeutic potential with BS along with activities like immunomodulatory and chemopreventive. Daucosterol is used as a natural effective component in chemotherapeutic drugs applied in cancer treatment (Zeng et al. 2017).

Triterpenoids

Terpenoids are a class of chemical compounds composed of Isoprene units, which is a 5-carbon compound, having the chemical formula, C5H8. Depending on the isoprene unit, terpenoids are classified into different categories. Among them, the triterpenoid is made up of 6 Isoprene units, with its chemical formula C30H48. Their biosynthesis, in animals and plants, is by Melovonic Acid Pathway (isoprenoid pathway) by the cyclization and produce beta oleane. The triterpenoid backbone then makes various modifications to redox substitution and glycosylation to produce further compounds (Zhou et al. 2017). They play an important role in plant growth and development. Moreover, they also play a major role in combating fungal pathogens and in conferring selective advantages, for example by suppressing the growth of neighboring plants or by protecting against pests, pathogens, and stress. They may also have subtle physiological roles in plants, which are yet uncharacterized (Cárdenas et al. 2019).

Various pharmacological actions, such as anticancer activity, are related to the presence of hydroxyl groups in certain pentacyclic triterpenoids. Recemosol, a polyhydroxy triterpenoid, has demonstrated substantial anticancer activity against human liver cancer (HepG2), human cervical cancer (HT-3), and normal human carcinoma (HeLa) cells (Kemboi et al. 2020). In addition, triterpenoids have been strongly linked to anti-inflammatory properties and drug resistance mitigation. It effectively inhibits NF-B activation, induces apoptosis, and suppresses inflammation, hence playing a crucial role in anti-inflammatory disorders (Huang et al. 2018). Furthermore, triterpenoids have played a significant role in breast cancer chemoprevention and treatment. Breast cancer continues to be a leading cause of death worldwide, and its restricted treatment choices have exacerbated the situation. Recent research has focused on triterpenoids and numerous human malignancies, including breast cancer. It has been proposed that triterpenoids, which disrupt and eliminate the STAT3 pathway, a main intrinsic mechanism for cancer inflammation, play a significant role in breast cancer treatment (Ghante & Jamkhande 2019).

Alpha-amyrin

α-Amyrin, a pentacyclic triterpenoid produced from plants, possesses numerous essential physiological and pharmacological properties. Gout is a common kind of inflammatory arthritis that affects a single joint at a time and causes inflammation, itching, and redness in the affected area. Gout disrupts a series of enzymatic pathways, and xanthine oxidase is one of them. It is a crucial enzyme that catalyzes the oxidation of hypoxanthine to xanthine to uric acid by the generation of reactive oxygen species. Hyperuricemia is the underlying cause of gout and is caused by an excess or deficiency of uric acid excretion (Battelli et al. 2018).

Recently, α-Amyrin was isolated by gas chromatography-mass spectrometry, electrospray ionization-mass spectrometry (ESI–MS), and nuclear magnetic resonance (NMR) and its anti-inflammatory activity against Gout disease was analyzed. The compound exhibited a high potential for preventing gout by inhibiting xanthine oxidase (XO) (IC50 = 258.22 µg/mL). In addition, another key enzyme in skin hyperpigmentation, tyrosinase, was also suppressed by the α-amyrin (Viet et al. 2021). Additionally, oxidative stress, an important physiological process, is involved in many chronic diseases such as cancer and diabetes. Oxidative stress can also induce an inflammatory process in chronic diseases and α-Amyrin has also shown antioxidant properties against such diseases. Additionally, α-Amyrin has been assessed for its anti-inflammatory and gastroprotective effects. Its effects on stomach ulcers and inflammation were examined in one study. The chemical effectively prevented stomach damage caused by ethanol or acidified ethanol in mice. Without affecting stomach secretory volume, the chemical considerably reduced overall acidity (Oliveira et al. 2004).α-Amyrin has also shown anti-fungal and anti-bacterial activity. The pervasiveness of antibiotic resistance of gram-negative strains is of main concern, especially for immunodeficient people. Hence, it is important to find new potential agents to combat these MDR strains (Nocedo-Mena et al. 2019). In addition, the emergence of new antifungal compounds is crucial since fungal cells are physically and metabolically like human cells, raising the risk of toxicity and harmful consequences of antifungal medications (Johann et al. 2007).

In addition, SARS-CoV-2 is the pathogenic agent of COVID-19, which has caused over 6 million deaths globally. Therefore, it is crucial to investigate and assess molecules with antiviral activity. The antiviral effect of α-Amyrin against spike glycoprotein and ACE2 receptor of SARS-CoV-2 was studied in one study (Maurya et al. 2020). According to the results, α-Amyrin was among the molecules with the highest affinity for spike glycoprotein and ACE2 receptors. The target prediction research found that this component targets the apoptotic pathway, nuclear transport, protein kinase B signaling, control of ion transport, and membrane protein proteolysis pathways, which are disrupted in this disease (Maurya et al. 2020).

Ursolic acid

Ursolic acid is likewise a pentacyclic triterpenoid, generated from α-amyrin, with multiple medicinal potentials. Protozoa consist of a vast array of unicellular organisms that can infect humans via numerous human-to-human or fecal-to-oral transmission routes. Protozoan diseases such as malaria, Chagas disease, and African trypanosomiasis, among others, are the leading causes of illness and mortality on a global scale. To solve this issue, it is required to develop new chemicals. Recently, it has been shown that Ursolic acid (ULA) has anti-protozoan action. ULA inhibits Leishmania species, the protozoan responsible for the tropical disease leishmaniasis, to a large degree. (Son & Lee 2020).

Neurodegenerative diseases (Parkinson’s and Alzheimer’s) are linked with neuro-inflammation—a complex process, regulated by microglia and astrocytes. These cells produce certain pro-inflammatory factors such as tumor necrosis factor-alpha (TNF-α), which promote inflammation by the activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways and trigger apoptotic processes. It has been estimated that the administration of Ursolic Acid downregulates the NF-κB pathway (Gudoityte et al. 2021). Multiple biological functions are performed by Ursolic Acid, including anti-oxidation, anti-cancer, anti-inflammatory, and hepatoprotection. Recent research studied the effects of ursolic acid on the development of nonalcoholic fatty liver disease (NAFLD). In-vitro and in-vivo analyses revealed a reduction in liver weight, serum ALT/AST levels, and palmitic acid, increasing lipid β-oxidation and reducing hepatic endoplasmic reticulum (ER) stress (Li et al. 2015).

Other compounds

The following compounds are derivatives of one of the above-discussed categories and besides Chimaphilin and methyl salicylate are the most important in terms of therapeutic and industrial potential.

5-hydroxymethylfurfural

It is a saccharide-derivative purified using a Sephadex LH-20 column by eluting with 100% methanol (Yu et al. (2021a, 2021b)). It has antibacterial, antiviral, anti-inflammatory, antifungal, anti-tumor, anti-hyperglycemic, analgesic, and anticonvulsant activities leading to its cardiovascular potential. The furan ring upon condensation with aldehydes and ketones gives secondary and tertiary alcohols, which further react with furan to form oligomers. Its furfural component is important in oil refining, it is also used as a bonding agent in grinding and abrasive wheels, in pharmaceuticals, and in the manufacture of phenolic resins. With the methyl group, the furfuran ring acts as a solvent and monomer; usually reacting with further compounds to form derivatives of furfurals. With the hydroxyl group, furfurals can be exploited to produce biomass-derived fuels and polymers by understanding the metabolism pathways (Yang et al. 2013).

Zhang et al. (2021) inhibit endoplasmic reticulum stress and NLRP3 inflammasome activation leading to the alleviation of lung injury. Aresta et al. (2019) have also worked on the two moieties of 5-HMF bearing the potential of the oxidation process and have exploited them to achieve microalgal components. Furthermore, many researchers have worked on the up-gradation of 5-HMF, and one of these studies (Yang et al. 2013) has worked on the up-gradation of 5-HMF to develop biorefineries through catalytic production of 5-HMF & up-gradation. Despite its profound potential for therapeutic and industrial applications, 5-HMF is not being exploited much because of its irritant potential, and until science does not find a way to get around this problem, all the work done on 5-HMF is, but theoretical.

Isohomoarbutin

Isohomoarbutin is a phenolic glycoside, a characteristic component of this genus. It is optically active and is readily hydrolyzed (Jiang et al. 2017). It is extracted using RP CC by gradient elution with methanolic water (Yu et al. 2021a, 2021b). The hydrolysis capacity of isohomoarbutin enables it to have almost 55 derivatives.

The first derivative is salireposide with hydrogens at the first four alkyl groups and OB1 at the R5 position. It has a high scavenging ability and is active against poliomyelitis and semliki forest virus. Furthermore, it is known to inhibit thymidine phosphorylases (Xu et al. 2015). The second derivative curculigoside has hydrogens at the first four alkyl groups’ position and B2 at the R5 position, where B5 = 4-hydroxybenzoyl. These compounds have distinct antioxidative and scavenging potential (Xu et al. 2015). The third derivative is xylosmin with B1 at the R1 and R4 positions, hydrogens at the R2 and R3 positions, and B13 at the R5 position, which has NS5 polymerase inhibitory activities against moderate dengue virus. Lastly, symponoside has hydrogens at the R1, R3, and R4 positions, B10 at the R2 position, and OB1 at the R5 position (Xu et al. 2015; Jiang et al. 2017).

Methyl salicylate

It is an aromatic ester and exists in a clear, colorless, and prismatic liquid state. Methyl salicylate is the essential oil of the C. umbellata plant. It is soluble in aromatic & aliphatic alcohols along with esters, is very stable in air, and does not hydrolyze in the presence of moisture. However, the compound is unstable when it encounters iron salts and produces pink-red discoloration. It has an organoleptic nature making it controversial for beverages except in North America. South America is one example of the controversies faced by methyl salicylate in beverage use. On the other hand, 50% of North America’s toothpaste flavor is methyl-salicylate based (Clark IV 1999).

Methyl salicylate has many health benefits including its potential against pain, gout, eye diseases, fever, and warts. What distinguishes this essential oil from all other oils is that it has never been reported in any animal flesh, fish, or crustaceans. Even in plants, it is restricted to some plants and is not produced in others, especially citrus fruits. Another idiosyncrasy of methyl salicylate is that no substitutes of methyl salicylate are available in the market, since no other compound has the compound’s profile (green, herbal, minty) (Clark IV 1999). It is well known that the first member of any chemical series has unique properties that are difficult to match, the same is true for methyl salicylate (Clark IV 1999). The odour of the oil faints as we increase the C number (for instance, ethyl, propyl, butyl) and the aromatic function and fragrance also decrease with a higher molecular weight, making methyl salicylate one of a kind.

Methyl salicylate is known as an inducer of plant defense against pathogens and certain herbivores. It is present in trace amounts in plants, which increases its market value. Currently, 7 industries across the globe produce methyl salicylate, and only one of these, Robertet, France, extracts it naturally from its plant species (Clark IV 1999), making the natural extraction of methyl salicylate a great start-up project. Along with its organoleptic properties, the irritant status of methyl salicylate contributes to its controversial market and manufacturing status. More research must be done to somehow counter these issues.

Chimaphilin

Chimaphilin (Fig. 4) is a member of 1,4-naphthoquinones and yellow needle crystal in appearance. Naphthoquinones in plants are known for their role in plant-plant, plant–microbe, and plant–insect interactions. Chimaphilin is a biologically active compound, and its IUPAC name is 2,7-dimethylnaphthalene-1,4-dione. It is one of the major active phytoconstituent of C. umbellate, extracted from various species of Chimaphila and Pyrola (Hausen & Schiedermair 1988). It is produced in the HGA/MVA pathway (also referred to as the toluhydroquinone or toluquinol pathway) in those plants but very little has been known about the metabolism of this pathway (Widhalm & Rhodes 2016).

Fig. 4.

Fig. 4

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Chimaphilin acts as an anti-oxidant, anti-cancer, and anti-fungal agent

Chimaphila umbellate has been used for its antifungal activity by the First Nations Peoples of eastern Canada. Galván et al. (2008) found that it was chimaphilin that acted as an antifungal agent against dandruff-causing fungi. They identified this compound as chimaphilin by 1H and 13C NMR spectroscopic analysis and Electron Impact Mass Spectrometry (EIMS) also showed 186 g/mol of molecular weight. All this data was consistent with the previous literature on this compound. Gene Deletion Array (GDA) of Saccharomyces cerevisiae mutants indicated that chimaphilin interferes with the cell wall, transcription, and mitochondrial functions. Results also showed that chimaphilin has a wide range of antifungal activities and that includes activities against fungal pathogens in humans. What makes it more interesting is that its modes of action are not known (Galván et al. 2008). The antioxidant activity of C. umbellate extract is mostly because of quercetin and isoquercetin but chimaphilin may also play a role as an antioxidant because other plant naphthoquinones like shikonin and plumbagin have shown significant antioxidant activities (Galván et al. 2008).

Chimaphilin's highly efficient anti-proliferative and anti-cancer activities have been studied in a variety of cancer cells including MCF-7 cells (Ma et al. 2014) and human osteosarcoma cells (Daqian et al. 2015). In human breast cancer, MCF-7 cells were initially examined, as well as the underlying processes to find out the anticancer efficacy of chimaphilin and it has been shown to reduce MCF-7 cell viability in a concentration-dependent manner. Chimaphilin triggered the production of reactive oxygen species and disruption of the mitochondrial membrane resulting in induced apoptosis (Ma et al. 2014). Anticancer drug cross-resistance is an unsolvable dilemma in the treatment of osteosarcoma. Drug resistance has proven to be a stumbling obstacle in the pursuit of higher cure rates. Tumor cells develop resistance to chemotherapeutic medicines because of this. But in a time and dose-dependent way, Chimaphilin reduced the development of both drug-sensitive and drug-resistant osteosarcoma cell lines (Daqian et al. 2015).

Issues regarding the utilization of Chimaphila umbellata

The first reason why C. umbellata is under-explored lies in its conservation status. According to Lundell et al. (2015), Sweden saw an exponential decrease in the population of C. umbellata in the past 10 years (2005–2015). Apart from Sydney, the plant is in the vulnerable condition in Norway and other areas of Europe, extinct in France and Switzerland, critical in the Czech Republic, and endangered in Slovakia, Hungary, Ukraine, and Germany (Allen et al. 2014). The same source (Allen et al. 2014) weighs in on the threats faced by the plant. These include soil disturbance, instant fires in dry seasons, over-harvesting, a cover of competitive species, disturbance in soil nitrogen content, habitat destruction due to tourism, and constant shade.

Although many conservation policies are in place at pan-European and international levels, the utilization of C. umbellata is still not preferred, at least until the plant is out of critical or vulnerable status. The pan-European policies in place include the Bern convention, the KU habitats directive, the KU wildlife trade regulation, and the 2020 strategy for biodiversity. The international-level policies include the conservation of biological diversity, the Nagoya protocol, CITES, and the convention on Medicine plants by WHO (Allen et al. 2014).

The biotechnological perspective

The conventions discussed in the previous section include conventional approaches. With an increasing biotechnological insight, the conservation of C. umbellata can be enhanced through targeted management of habitats, reintroduction of the plant in the moderate disturbance regimes, proper harvesting strategies, updated assessment of conservation status, regulated access to genetic resources, and soil analyses and modification (Kikuchi et al. 2021). The modern and molecular strategies include micro-propagation of the endangered plant in-vitro, which comes with its challenges as discussed in the next section; mycorrhization of the plant and its associated fungi, genetic transformation of C. umbellata improving its growth conditions so it may withstand competition and might not die because of its slowed germination (Figura et al. 2018). DNA banks and cryopreservation are the newest approaches in combating the scarcity of medicinally important resources.

Based on the DNA sequencing reads acquired via 454 pyrosequencing, primers amplifying 16 microsatellite loci were created for the endangered semi-shrub Chimaphila umbellata, which is only found rarely in the Japanese Archipelago. This study was carried out by Kikuchi et al. (2017). The populations that were sampled from the Hokkaido District and the Tohoku District both had polymorphism versions of these 16 loci; the average number of alleles was 3.31 and 3.44, respectively, and the mean predicted heterozygosity was 0.42 and 0.44, respectively. These genetic markers were not related to one another in any way, nor did they have any null alleles. Amplification using these primers was also tried in the congeneric species C. japonica; however, only three of them were able to successfully amplify the DNA of the species (Kikuchi et al. 2017). These markers are useful for analyzing the genetic diversity of C. umbellata populations as well as the genetic divergence between those populations.

Critical analysis of the issues

Based on our extensive study and apart from the obvious conservation status challenges of C. umbellata, we have identified eight major challenges of working with this plant. These are discussed as follows:

  1. Phytochemical concentration: all the key phytochemicals in C. umbellata are found in trace amounts. Although this increases their market value, extracting them and further down-streaming of these phytochemicals is a major challenge faced by the researchers.

  2. Metabolic pathways: although a putative biochemical pathway for the major phytochemical production is put forward by Widhalm & Rhodes (2016) in Nature, it lacks the key pathways and intermediates leading to the production of certain phytochemicals.

  3. Commercialization: the controversial status of C. umbellata and its phytochemicals is a big hurdle in the commercialization of its products. Furthermore, the irritant, genotoxic, and biohazard status of many of its phytochemicals is another problem in its commercialization.

  4. Mycorrhizal association: little is known about the mycorrhizal association of C. umbellata making it harder to grow in-vitro and other than in its natural environments.

  5. In-vitro cultivation: as discussed in the previous sections, no single media has been optimized to date to cultivate C. umbellata.

  6. Lack of literature: A huge gap is found in C. umbellata’s literature from 2008 to 2017 because of its conservation status. This gap has resulted in problems like missing metabolic pathways and research on applications of C. umbellata’s products and phytochemicals.

  7. Slow seed germination: as discussed earlier, one of the primary reasons for C. umbellata extinction is its slow germination, leading to the exhaustion of resources before the plant can utilize them.

  8. ADMET properties: the lack of research on the plant, especially in an age where biotechnological and bio-informational approaches are at their peak, has led to gaps regarding several medicinally important characteristics.

Recommendations and conclusion

As per the issues discussed above, the following recommendations, if implemented, can allow us to harvest C. umbellata to its potential.

  1. We need to focus on media optimization and work to produce in-vitro cultures of C. umbellata

  2. The potential of C. umbellata to produce nanoparticles and the capacity of these NPs as carriers & adjuvants must be studied

  3. We should identify and optimize elicitation factors and pathway precursors or intermediates to enhance the phytochemical content

  4. The ADMET analysis along with other essential regulatory analyses to commercialize the plant’s compounds must be undertaken.

The critical role of bioinformatics

Bioinformatics has reduced the research and development time by half. The online and offline software available for analyses allows researchers to make predictions about their required conditions and characteristics in real time. In-vitro and in-vivo analyses are then performed based on positive predictions of the in-silico models. In terms of C. umbellata, bioinformatics can be used to counter the metabolism-related challenges using pathway identification software like Cytoscape, STRING, and KEGG, identify lead compounds to replace or modulate compounds like methyl salicylate that do not carry the irritant quality. Furthermore, QSAR and ADMET software can be employed to identify and optimize these compounds, and pharmacophore modeling can be used to access and define new constraints to upgrade the phytochemicals for their industrial and medicinal use.The merger of transcriptomics and metabolomics data can lead to the identification of key metabolic pathways (Gu et al. 2017; Xiao et al. 2022), tools and repositories of herbal genomics along with in-silico drug discovery mechanisms can be explored (Chakraborty 2018), metabolic engineering and elicitation to enhance the phytochemical synthesis can be assessed (Mora-Vásquez et al. 2022), and metabolomics and next-generation sequencing data can be integrated to elucidate the pathways of natural product metabolism (Scossa et al. 2018).

Funding

The authors did not receive support from any organization for the submitted work.

Declarations

Conflict of interests

The authors have no relevant financial or non-financial interests to disclose.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Urooj Ali, Email: msurooj9@gmail.com.

Muhammad Mustajab Khan, Email: gfx.mustajab@gmail.com.

Naveera Khan, Email: naveerakhan@bs.qau.edu.pk.

Rida tul Haya, Email: Ridatulhaya20@gmail.com.

Muhammad Usama Asghar, Email: Usamakhan6922@gmail.com.

Bilal Haider Abbasi, Email: Bhabbasi@qau.edu.pk.

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