Insights into nuclear DNA content, hydrogen peroxide and antioxidative enzyme activities during transverse thin cell layer organogenesis and ex vitro acclimatization of Malaxis wallichii, a threatened medicinal orchid.

Malaxis wallichii (Lindl.) Deb, a small, perennial, monopodial, terrestrial orchid, is endemic to tropical Himalayas at an altitude of 1200-2000 m asl. The pseudobulbs are important ingredients of century old drug 'Ashtavarga' and a polyherbal energetic tonic 'Chyavanprash'. An efficient genetically stable in vitro propagation protocol using transverse thin cell layer culture system was established for M. wallichii. In the present report, meta-topolin alone proved to be three times more beneficial compared to other routinely used cytokinins in inducing highest number of shoot buds, plant height and growth of regenerated shoots. The highest regeneration frequency (89%) along with maximum number of adventitious shoots per explant (22.5 ± 0.6) was observed in MS medium supplemented with 1.0 mg/l meta-topolin and 0.5 mg/l α-naphthalene acetic acid. Highest rooting frequency with highest number of roots (8.66 ± 0.3) was achieved in half-strength MS medium fortified with 1.0 mg/l indole acetic acid. Clonal stability of in vitro-derived plantlets was evaluated and compared to donor plant using intron splice junction (ISJ) markers and flow cytometry. ISJ markers revealed 4.76% clonal variability indicating high degree of genetic stability amongst the in vitro-derived regenerants. The nuclear DNA content of M. wallichii (2n) was found to be 2C = 2.760 ± 0.02 pg and therefore, 1349.64 Mbp (1C). Flow cytometry analysis of actively growing young and mature leaves from donor as well as in vitro-derived plantlets revealed presence of three peaks corresponding to 2C, 4C and 8C, while 2C was the most abundant. In the present investigation, there was no significant difference in the 2C DNA content between the mother and in vitro-derived plants; however, the frequency of endopolyploid cells varied in young and adult plants. An increased H2O2 content as well as lipid peroxidation activities were observed during early stages of acclimatization which declined afterwards. The enhanced activities of antioxidant enzymes like superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase in acclimatized plantlets as compared to in vitro-grown ones revealed their active involvement in growth and development against oxidative stress under external adverse environment.

Secondary metabolite profiling, cytotoxicity, anti-inflammatory potential and in vitro inhibitory activities of Nardostachys jatamansi on key enzymes linked to hyperglycemia, hypertension and cognitive disorders.

ETHNOPHARMACOLOGICAL RELEVANCE: Nardostachys jatamansi (D. Don) DC., 'Spikenard' or 'Jatamansi', a highly valued, aromatic herb from alpine Himalayas has a long history of use as ethnomedicine and dietary supplements in Ayurveda, Unani and Chinese system of medicine since Vedic ages (1000-800 BC). In Ayurveda and traditional system of medicine, the species is used as stimulant, sedative, brain tonic or mind rejuvenator, antidiabetic, cardio tonic, and in the treatment of various neurological disorders such as insomnia, epilepsy, hysteria, anxiety and depression. It is considered as Sattvic herb in Ayurveda and is now commercially marketed either as single or poly-herbal formulations by many companies in national and international markets. AIM OF THE STUDY: The species has become threatened in its natural habitats due to over exploitation and illegal trade of its rhizomes for drug preparation in herbal and pharmaceutical industries. Considering the increasing demand and tremendous medicinal importance of this threatened plant species, a detailed study was undertaken to evaluate its antioxidant potential, secondary metabolite profiling, cytotoxicity, anti-inflammatory potential and in vitro enzyme inhibitory activities on key enzymes linked to hyperglycemia, hypertension and cognitive disorders in different plant parts of wild and in vitro-raised plants with respect to different solvent systems for its sustainable utilization. MATERIALS AND METHODS: Anti-cholinesterase activity of leaves and rhizome of wild and cultured plant extracts was investigated against both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) enzymes. In vitro anti-hyperglycemic (α-amylase and PTP1B), anti-hypertensive (angiotensin-converting enzyme), anti-tyrosinase and anti-inflammatory potential (5-lipoxygenase and hyaluronidase) of different plant parts of wild and in vitro-raised plants with respect to different solvent systems were also evaluated. In vitro cytotoxic effect of rootstock extracts of wild and in vitro-derived plants were against cancer (HCT-116, MCF-7 and OE33) and two normal (HEK and MEF) cell lines. Secondary metabolite profiling of rhizome segments of wild and in vitro-derived plants was carried out by quantitative gas chromatography-mass spectrometry (GC-MS). RESULTS: In vitro-raised plantlets showed comparative higher yield of various secondary metabolites with a significantly high antioxidant activity as compared to the wild plants. Methanolic rootstock extracts of both wild and in vitro-derived plants of N. jatamansi exhibited significant AChE (IC50 36.46 ±â€¯2.1 and 31.18 ±â€¯2.6 µg/ml, respectively) and BuChE (IC50 64.6 ±â€¯3.5 and 60.12 ±â€¯3.6 µg/ml, respectively) inhibitory potential as compared to standard inhibitor galanthamine (IC50 0.94 ±â€¯0.03 and 4.45 ±â€¯0.5 µg/ml). Methanolic rootstock extract of in vitro-derived plants showed significant α-amylase (IC50 90.69 ±â€¯2.1 µg/ml), PTP1B (IC50 24.56 ±â€¯0.8 µg/ml), angiotensin-converting enzyme (IC50 42.5 ±â€¯3.6 µg/ml) and tyrosinase (IC50 168.12 ±â€¯3.6 µg/ml) inhibitory potential as compared to standard acarbose (IC50 52.36 ±â€¯3.1 µg/ml), ursolic acid (IC50 5.24 ±â€¯0.8 µg/ml), captopril (IC50 32.36 ±â€¯2.5 µg/ml) and kojic acid (IC50 = 54.44 ±â€¯2.3 µg/ml). Both the methanolic rootstock and leaf extracts of tissue culture-derived plants exhibited promising anti-5-LOX and anti-hyaluronidase activities against the known inhibitor of 5-LOX and hyaluronidase. Furthermore, methanolic rootstock extracts of both wild and in vitro-derived plants exhibited promising cytotoxic effects to HCT-116, MCF-7 and OE33 cell lines as compared to the normal HEK and MEF after 12 h of treatment. Secondary metabolite profiling of wild and in vitro-derived plants by quantitative GC-MS analysis revealed the presence of different classes of terpenoids and phenolic acids might be responsible for its effective biological activities. CONCLUSION: In vitro-derived plants revealed a substantial anti-cholinesterases, anti-hyperglycemic anti-inflammatory, anti-hypertensive and anti-tyrosinase potential with higher yield of various bioactive metabolites and significantly higher antioxidant activity which substantially explain medicinal importance of N. jatamansi in traditional medicine, used for centuries in different Ayurvedic formulations. The present findings suggest that cultured plants could be a promising alternative for the production of bioactive metabolites with comparative biological activities to the wild plants.

Studies on secondary metabolite profiling, anti-inflammatory potential, in vitro photoprotective and skin-aging related enzyme inhibitory activities of Malaxis acuminata, a threatened orchid of nutraceutical importance.

Malaxis acuminata D. Don., a small, terrestrial orchid, is endemic to tropical Himalayas at an altitude of 1200-2000m asl. The dried pseudobulbs are important ingredients of century old ayurvedic drug 'Ashtavarga' and a polyherbal immune-booster nutraceutical 'Chyavanprash', known to restore vigour, vitality and youthfulness. Considering tremendous medicinal importance of this threatened orchid species, a detailed study was undertaken for the first time to address its antioxidant potential, secondary metabolite contents and biological activities against skin-aging related enzymes (anti-collagenase, anti-elastase, anti-tyrosinase and xanthine oxidase) and anti-inflammatory activity (5-lipoxygenase and hyaluronidase) in different plant parts of wild and in vitro-derived plants of M. acuminata. Methanolic leaf and stem extracts were further evaluated for in vitro photoprotective activity against UV-B and UV-A radiations. Furthermore, secondary metabolite profiling of various plant parts was carried out by Gas Chromatography Mass Spectrometry (GC-MS). A significantly higher antioxidant potential (DPPH, metal chelating and ABTS•+) with a comparative higher yield of secondary metabolites was observed in in vitro-derived plantlets as compared to the wild plants. Among various solvent systems used, methanolic leaf and stem extracts showed promising inhibitory activity against major skin aging-related enzymes and anti-inflammatory potential. Methanolic leaf and stem extracts of both wild and in vitro-derived plants showed promising photoprotective activity against UV-B and UV-A radiations in vitro with comparatively higher sun protection factor (SPF). Furthermore, GC-MS analysis of methanolic extracts of leaves and stems of wild as well as in vitro-derived plantlets revealed presence of many bioactive metabolites such as, dietary fatty acids, α-hydroxy acids, phenolic acids, sterols, amino acids, sugars and glycosides which substantially explain the use of M. acuminata as one of the potential rejuvenator and anti-aging ingredient in many Ayurvedic formulations.