Journal of Innovation in Applied Research (eISSN: 2581-4206)

Introduction

Plants have been employed as a primary natural source of alternative medicines and have played an essential part in human lives from ancient times. The ancient practise of using therapeutic plants has evolved into a very valuable industry in the worldwide market, resulting in the release of a vast number of herbal medicines [1].Curculigo orchioides Gaertn. belonging to Hypoxidaceae family, often known as black musli, is a significant monotypic taxon of India.It is a major Rasayana drug in Ayurvedic system. The ayurvedic medicament is extracted from the mucilaginous tuberous roots of Curculigo orchioides [2]. It is referred by numerous vernacular names, Talmuli,Talusain West Bengal, while in Tamil Nadu it is known as Nilappanai.In Karnataka, Kerela and Andhra Pradesh it is known as Nelatigade, Nelappana and Nelatadi respectively [3]. The herb C.orchioides is known for its aphrodisiac quality thus is used in traditional Chinese medicinal drugs to cure impotence [4]. Many different properties are also found like diuretic, antiulcer and tonic properties. Diseases like asthma, jaundice, venereal and urinary are also cured with the help of this drug [5]. Antioxidant, hepatoprotective, immunomodulatory and anticancer properties have also been found in this plant and thus has been intensively researched [6,7]. This perennial herb is found all over India, notably in subtropical climate of Western ghats and Himalayan region, up to 2250 m altitudes. The plant has also been reported in Assam, West Bengal, Bihar, Uttar Pradesh, Himachal Pradesh, Gujarat, Andhra Pradesh, Karnataka, Kerela and Tamil Nadu [8]. The availability of C. Orchioides in India is shown in Figure 1.

The drugs are extensively used to study phytochemicals and pharmacological activities [9,10,11]. C. Orchioides roots are widely used in pharmaceutical enterprises to make medications, and a significant number of roots are harvested from woods by local residents and tribal people. It commands a high market price. The natural populations of this plant have been regarded low due to merciless economic exploitation and habitat loss, as well as poor seed laying and germination [12]. The plant is not grown on a wide scale in the nation, although there are some occasional cultivation and tissue culture techniques available for in vitro culture and fast multiplication which have been discussed ahead.

Picture showing the occurance of Curculigo orchioides in shaded areas.>

The basic biology of Curculigo orchioides

C. orchioidesis an ever-lasting herb with a height of 30 cm. The rhizomes are erect, and thick with creeping slender stolon. The leaves are often 4–7 cm long; the petiole is 30–80 cm long; the berries are white, sub-globose, and beakless, and the seeds are black with irregular stripes. The plant originated in Asia's remote woods. On sodden-rich soil, the plant is circulated in fields. It demonstrates prostate growth. Appearing in portions of India from close to the ocean level of about up to 2100 m height, especially in the rock holes and laterite soil [13]. Flower blossoms as the year progresses, and is light yellow in colour, sexually open, sessile, customary, and 1.2 cm. Perianthhassix lobes, which are yellow in colour [0.5-1 x 0.20-0.30 cm], stamens are 6 in number, filaments 2mm, stringy, anthers 2.0 mm, 3 celled ovaries, elongated to 4.0 mm. There are various ovules per cell, style is 2.0 mm, shame 3.0, and projections prolong [14]. Fruit is capsulated, elongated glabrescent with a slim mouth and springy ‘septa’, 1.50-2.0cm in length 8.0mm broad; Seeds are 8 in number, ‘globose’, 1.0-2.0mm, dark, angled, profoundly scored in wavy lines [15,16]. The plant of C. orchioidesis represented in Figure 2.

Figure 2 Plant of Curculigo orchioides

Phyto-chemistry

The chemical constituents of C. orchioideshave also been investigated. This summary allows an understanding of the general chemical information and the bioactive constituents that have been discovered. The compounds include phenols and phenolic glycosides [1–17], lignans and lignan glycosides [18-20], triterpenes and triterpenoid glycosides [21-41], flavones [42-43], alkaloids[44-47], aliphatic compounds [49-54], polysaccharides [55-56] and other types of compounds [57]. COPb-1 and COPf-1 are two water-soluble polysaccharides from Curculigo orchioides Gaertn and their molecular weights are 2.6×10⁶ Da and 2.2×10⁶ Da, respectively. COPb-1 is composed of glucose, fructose and xylose, and COPf-1 consists of stachyose, glucuronic acid and galacturonic acid [17]. The compounds that have been isolated from the plant have been documented and listed in Table 1.

Tissue culture techniques for cultivation

The genetic diversity of medicinal plants across the world is being threatened at an alarming rate due to destructive harvesting techniques and over-harvesting for medicine manufacture. Solid procedures for the mass propagation of endangered species are extremely appealing for meeting the needs of professional plantings and plant merchants while also re-establishing the plants to their natural environment [42]. C.orchioides is in high demand due to its numerous applications; nevertheless, its availability is unpredictable and insufficient. [43]. this plant species is presently threatened owing to habitat reduction in natural habitats that sustain vegetation. The following are the major contributing factors:

1. Cattle ranching and leaf litter amassing have resulted in massive denudation of the forest floor.
2. Extraction from the forest for tuberous roots, which are highly valued in the market for their metabolic boosting principles and aphrodisiac compositions.
3. A lack of seed establishment and germination
4. A significant prevalence of viral and bacterial rhizome infections
5. Many tribal people consume the rhizome as edible flour.
6. Usage of the plant as a safe substitute for Safed Musli [44]

C.orchioides has also been included to the list of endangered plants by the Department of Biotechnology, Ministry of Science and Technology, New Delhi [45]. The progressive loss of this species' population necessitates the introduction of conservation initiatives to assure continuous and abundant supply by maintaining a balanced cycle of harvest and rejuvenation [46]. Keeping in mind the aforementioned facts, namely the progressive reduction of this endangered species, plant tissue culture has evolved into an effective technique for developing micropropagation systems for such plants [47].Thus, we present an overview of previous researchers' in vitro propagation protocols for mass multiplication and conservation of this multipotential medicinal plant: C. orchioides, using different explants on different types of media, at different concentrations and combinations ofgrowth regulators.

Shoot tip Culture

Through apical meristem culture an effective approach for in vitro clonal growth of C.orchioidesGaertn. was devised. Murashige and Skoog [MS] basal media supplemented with 1.5 mg/l6-benzyladenine [BA], 100 mg/l adenine sulphate [Ads], and 3% sucrose was used to produce multiple shoots from apical meristems.The addition of indole-3-butyric acid [IBA] or indole-3-acetic acid [IAA] to the culture media aided in the production of numerous shoots [48].

Callus induction

The responses of rhizome discs from proximal to distal end were evaluated on MS basal media supplemented with varied quantities and combinations of auxins and cytokinins for callus induction; luxuriant callus was induced from the proximal end of the shoot axis rather than the distal end. Based on colour and texture, two types of callus were identified. Type I was induced on MS media which was supplemented with 2,4-D or NAA alone or NAA in combination with cytokinins. While Type II callus was grown on media with BAP or Kn in combination with 2,4-D [49].

Leaf culture

Various experiments have been performed for rapid propagation of the plant through leaf culture. C. orchioides leaf explants cultivated on an MS medium lacking cytokinins generated a limited number of plantlets that arose straight from the cut end of the midrib.Plantlets from rhizome explants required BA [50]. Direct inoculation of leaf pieces on MS medium supplemented with benzylaminopurine [BAP] [2-8 M /L] or thidiazuron [TDZ] [2- 8 M /L] alone or in combination with naphthaleneacetic acid [NAA] [0.5 and 1.0 M /L] produced low shoot induction in terms of percentage of response and number of shoots per explant.As a result, leaf explants were pre-treated with 15, 25, or 50 M/L TDZ for 6, 24, or 48 hours in order to improve shoot regeneration from cultured explants. Pre-treatment of explants with 15 mol/L TDZ for 24 h greatly increased the production of adventitious shoots, with the greatest response seen on MS medium supplemented with 6 M /L TDZ [51].

Root induction

A number of studies accomplished and reported rooting by moving micro shoots to half-strength MS medium with varying quantities of auxins. The shoots were rooted in media supplemented with either 0.54–5.37 M of 1-naphthaleneacetic acid [NAA], 0.57–5.71 M of indole-3-acetic acid [IAA], or 0.49–4.90 M of indole-3-butyric acid [IBA] [52].

Rhizome disc

Proximal rhizome discs outperform shoot tip and distal rhizome discs for high-frequency shoot bud development. It was discovered that 6-benzylaminopurine [BAP] and kinetin [Kn] [both at 1 mg/L] had a synergistic impact on the regeneration of shoot buds from the proximal rhizome disc [53].

Table 2 Tissue culture techniques of C. orchioides

Arbuscular mycorrhizal [AM] fungal inocula

The influence of three arbuscular mycorrhizal [AM] fungal inocula on post-transplantation performance of in vitro grown C. orchioides plantlets was observed.The three AM fungal inocula comprised of two monospecific cultures of Glomus geosporum and G. microcarpum and one crude consortium of AM fungal spores isolated from C. orchioides rhizosphere soil growing in natural environment.For the sustainable production and protection of this rare medicinal plant, the study advises using a mixed consortium of AM fungus rather than monospecific cultures [54]. The discussed tissue culture techniques have been listed in the Table 2.

Medicinal properties of Curculigo orchioides

Curculigo orchioides ethanol extracts have been proven to improve adaptive effects. These extracts may improve tolerance to extreme temperatures and hypoxia. They had sedative, anticonvulsant, androgen-like properties, as well as enhanced immunological activity in mice [55]. Methanol extracts of Curculigo orchioides rhizomes have also been shown to raise white blood cell counts, humoral antibody [HA] titer, and delayed type hypersensitivity [DTH] reaction in cyclophosphamide-treated immunocompromised mice.These findings suggest that methanol Curculigo orchioides extracts stimulate the immune system through mediating cells and humoral antibodies [56].

In both in vivo and in vitro experiments, Curculigo orchioides rhizome extracts demonstrated antiosteoporotic efficacy.. In ovariectomized rats, an ethanol extract of Curculigo orchioides rhizomes reduced bone loss in the trabecular bone of the tibia through regulating osteoprotegerin, the ratio of deoxypyridinoline crosslinks to creatinine, and tartrate-resistant acid phosphatase [TRAP] activity [57]. Curculigo orchioides rhizome methanol extract was shown to be significantly efficient in scavenging superoxide radicals and moderately effective in scavenging DPPH radicals, nitric oxide radicals, and inhibiting lipid peroxidation [58]. In rats treated with carbon tetrachloride, the methanol extract of Curculigo orchioides rhizomes increased food consumption and weight gain, decreased serum levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and gamma glutamyl transpeptidase, reduced serum levels of total protein and liver, and restored serum levels of total lipid, triglyceride, showcasing that the rhizome has hepatoprotective activity [59].

In isolated mouse peritoneal mast cells and mice exposed to compound 48/80-induced systemic anaphylaxis, an ethanol extract of the Curculigo orchioides rhizome significantly reduced mast cell degranulation [60]. Curculigo orchioides ethanol extract was reported to have relaxing action in isolated goat tracheal chain preparation and isolated guinea pig ileum preparation.Further research revealed that the extract had a strong protective effect against guinea pig bronchoconstriction, rat passive paw anaphylaxis, and haloperidol-induced catalepsy in mice.These findings suggest that Curculigo orchioides ethanol extract may be beneficial in treating asthma [61].

Curculigoside dramatically decreased N-methyl-D-aspartate-induced neuronal cell loss, the amount of apoptotic and necrotic cells, excitotoxicity, and intracellular reactive oxygen species [ROS] generation in cultured cortical neurons. Curculigoside's neuroprotective effects may be mediated via lowering apoptotic protein levels and decreasing intracellular ROS generation in cultured cortical neurons [62]. Recent research has linked nuclear factor-κB [NFκB] and high-mobility group box 1 [HMGB1] to the pathogenesis of cerebral ischemia. Curculigoside treatment of SH-SY5Y cells reduced oxygen-glucose deprivation-induced cytotoxicity and apoptosis, prevented TNF-α-induced NF-κB and IκB-α phosphorylation and also lowered HMGB1 expression [63].

Curculigo orchioides rhizome oil demonstrated significant antimicrobial activity against Bacillus anthracis, Bacillus subtilis, Salmonella pullorum, Salmonella newport, and Staphylococcus aureus, as well as fungal strains Fusarium moniliforme, Fusarium solani,

Table 3 Medicinal properties of C. orchioides

Aspergillus flavus, and Cladosporium spp. [64]. Curculigo orchioides rhizome gel formulations exhibited considerable anti-inflammatory effect against carrageen-induced rat paw edema [65]. In the Philippines, the rhizomes are used as a tonic, pectoral, diuretic, and aphrodisiac alone or in conjunction with carminative medications, and are put into a salve for itchy skin and other skin diseases [66]. A study indicates that Curculigo orchioidesGaertn fractions and compounds efficiently suppressed H2O2 induced oxidative stress by boosting antioxidant enzyme content and have cytotoxic potential on cancer cell lines HepG2, HeLa, and MCF-7 [67]. The various medicinal properties of Curculigo orchioideshas been listed in the Table 3.

Toxicology

As per Chinese Pharmacopoeia records, Curculigo orchioidesis poisonous and the clinical dosage suggested for adults is 3-9 g daily [68]. Curculigo orchioides' hepatotoxicity may be caused by a triterpenoid ketone, which reduced the viability of a human liver cell line. HL-7702. The dosages chosen for the toxicity trials of Curculigo orchioides [30 g/kg, 60 g/kg, and 120 g/kg] appear to be excessively high, and while no fatality was recorded, there were some adverse effects [69]. As a result, toxicity studies at lower levels are required to produce physiologically significant results. In general, taking Curculigo orchioides at the recommended clinical daily dose does not induce any severe side effects in people. However, taking Curculigo orchioides in high quantities over an extended length of time might produce cold sweats and numbness in the extremities.As a result, measures should be made to ensure the safe use of Curculigo orchioides; usage warnings have arisen in the medical literature. This is especially critical for the liver, kidneys, and reproductive systems [17].

Future prospects of Curculigo orchioides

A direct comparison of inhibitory potency and IC50 profiles of compounds demonstrated that chemicals from the plant [capsaicin and piperine] show stronger binding affinity with the provided antioxidant enzymes than the rest of the compounds. Thus, these chemicals may be more effective antioxidants in reducing oxidative stress by boosting the antioxidant enzyme defence system, which in turn activates anticancer enzymes. Nevertheless, additional research on the antioxidant and anticancer properties of the specific chemicals is required. Furthermore, additional research may be conducted on the pathways involved at the cellular level, which can provide a concise overview of the active cell machinery engaged in programmed cell death. Understanding the molecular mechanisms that drive apoptosis in response to anticancer medicines, as well as how cancer cells avoid apoptotic death, opens up new avenues for a more balanced approach to developing molecular-targeted cancer treatments [67]. Curculigo orchioides was also shown to lower hearing threshold shifts, central auditory function damage, and cochlear function deficits, indicating that it might be used as a possible therapeutic natural product for noise-induced hearing loss in mice [70].

Conclusions

We reviewed the known traditional usage of medicinal plants: Curculigo orchioides as well as studies on their phytochemistry, pharmacology, toxicity, and in-vitro propagation techniques in this study. It is on the verge of extinction and hence requires both ex-situ and in-situ conservation. Its cultivation is also necessary to suit industry needs. Furthermore, it is critical to discover whether pharmacological research on this plant is accessible to validate their traditional usage. Modern pharmacological investigations have investigated the traditional medicinal applications of Curculigo orchioides in the traditional medicine system. Indigenous, limited distribution, small population, accessibility in specific season, poor seed setting and germination have resulted in a fall in natural population as well as a big problem for exploitation/extraction. To address these issues, in vitro cultures might well be employed as an alternate approach for drug extraction and preservation.

Abbreviations

MS media Murashige and skoog media

BA Benzyl adenine

Ads Adenine sulphate

NAA Naphthaleneacetic acid

IAA Indole-3-acetic acid

IBA Indole-3-butyric acid

BAP Benzylaminopurine

Kn Kinetin

2,4-D 2,4-Dichlorophenoxyacetic acid

TDZ Thidiazuron

AM Arbuscular mycorrhizal

DTH Delayed type hypersensitivity

HA Humoral antibody

TRAP Tartrate-resistant acid phosphatase

ROS Reactive oxygen species

NFκB Nuclear factor-κB

HMGB1 High-mobility group box 1

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¹University Institute of Biotechnology, Biotechnology, Chandigarh University (CU), Punjab, India

²School of Biotechnology, Shoolini University of Biotechnology and Management Sciences, Bajhol, Solan, H.P., India