Comparative GC – MS Analysis of Bioactive Phytochemicals from Different Plant Parts and Callus of Leptadenia reticulata

Aim: The aim of this study is identification and comparative analysis of bioactive phytochemicals present in methanol and ethyl acetate extracts of different plant organs and callus of Leptadenia reticulata by GC–MS technique. Methodology: The shade dried plant samples (leaves stem and root) and callus produced from leaf of L. reticulata were powdered and then sequentially extracted in methanol and ethyl acetate solvents. Total eight extracts were prepared which were Methanolic Leaf Extract (MLE), Methanolic Stem Extract (MSE), Methanolic Root Extract (MRE), Methanolic Callus Extract (MCE), Ethyl Acetate Leaf Extract (EALE), Ethyl Acetate Root Extract (EARE), Ethyl Acetate Stem Extract (EASE) and Ethyl Acetate Callus Extract (EACE). Then, each of the extracts was further subjected to Gas Chromatography– Mass Spectrometry (GC–MS) analysis. Results: The GC–MS analysis of the eight extracts revealed the presence of 77 different types of high and low molecular weight phytochemicals and bioactive compounds in varying quantities. Some of the phytochemicals detected for first time in L. reticulata are γ-sitosterol, Campesterol, Pristane, Hexahydrofarnesol, Stearic acid, Arachidic acid, Coniferyl alcohol, n-Tetracosanol-1, Ascorbic acid 2,6-dihexadecanoate, (2S,3S)-3,7,4’-Trihydroxy-5-methoxy-6-methylflavanone etc. These chemical compounds are considered biologically active and pharmacologically important. Conclusion: This study gives a detailed comparison of detection and identification of various bioactive phytochemicals from different plant parts (leaves, stem and root) and callus of L. reticulata. This provides a basis for the biological and biochemical characterization of some newly detected biologically and pharmacologically important phytochemical components from this plant.


INTRODUCTION
Phytochemicals present in medicinal plants have been part of phytomedicines since ancient times.Medicinal plants play a vital role in the prevention and treatment of various diseases and largely contributing in all existing prevention strategies.Various plant-based medicines have already proved their potential efficacy and safety. 1 Now a days, synthetic drugs are broadly used but their extreme use may cause severe side effects in body which are sometimes more serious than that of disease itself.Hence, pharmaceutical companies are spending a lot of money and time on the plants with potential medicinal properties for the formulation of the natural drugs which are safe and effective. 2In developing countries all over the world, large number of people do not have access to synthetic drugs or they do not have potential to buy them, they still depend on traditional medicinal plants as they are cheaper and easily accessible.These reasons might account for the worldwide attention and use of medicinal plants.In 2013, WHO prepared and lunched 'WHO Traditional Medicine Strategy 2014-2023' which emphasized to join together traditional and complementary medicine to promote universal healthcare and to ensure the safety, quality and effectiveness of such medicines. 3pproximately 20% of the plants found in the world have undergone pharmaceutical or biological tests. 4lants have the ability to produce a number of compounds in the form of secondary metabolites that have diverse biological properties and they serve as active drugs against various diseases. 2,5Amount of these secondary metabolites varies from species to species and plant to plant depending on the age and variations in climates and ecological factors. 2 The phytochemical accumulation in plants has generated understanding for the production of desired bioactive compounds with the prospective to produce desired component.In many Pharmacognosy Journal, Vol 11, Issue 1, Jan-Feb, 2019 cases, crude extracts from medicinal plants are more biologically active than isolated compounds due to their synergistic effects. 6Extraction and characterization of numerous bioactive compounds from these green plant factories have given birth to various high activity profile drugs.A number of different solvent systems like water, methanol, ethanol, chloroform, methanol, ethyl acetate, petroleum ether etc. have been reported to play important role for extraction of secondary metabolites, such as tannins, terpenoids, steroids, alkaloids, flavanoids, phenols and quinines. 2eptadenia reticulata Wight and Arn.(Asclepiadaceae) is a well-known tonic and life giving drug with rejuvenating, restorative, antiabortificent and lactogenic properties.It is a perennial woody climber called as 'Jeevanti' in Ayurveda which means 'life-giver' because it works as a stimulant and claimed to prevent miscarriage. 7,8Jeevanti is used in Ayurveda since 4500 BC as a general body vigor provider.According to Atharva-Veda this plant promotes vitality and life. 7L. reticulata grows well in tropical and subtropical climate with moderate rainfall and relative humidity.This plant is also found in arid regions with sandy soil, low organic matter and very low rainfall. 9In India, L. reticulata is found in Sikkim, Karnataka, Rajasthan, Gujarat, Khasia hills, Nilgiris, Tamil Nadu, Laccadive Islands, Andhra Pradesh, Goa, Haryana, Kerala, Madhya Pradesh, Maharashtra, Orissa, Bihar, the sub-Himalayan tracts of Punjab, U.P. and Deccan Peninsula up to an altitude of 900 meters. 8,10part from India, it is distributed in the tropical and subtropical parts of Africa, Cambodia, Mauritius, Sri Lanka, Burma, Nepal, Madagascar, Malay Peninsula, the Philippines etc. 10,11 L. reticulata has many biological activities like antiabortifacient, 12 galactagogue/lactogenic/milk increasing effect, 13 anti-implantation, 14 antimicrobial, antioxidant, 15 antitumour, 16 immunomodulatory activity, 17 antiepileptic potential, 18 analgesic, 9 antipyretic, anti-inflammatory, 19 hepatoprotective, 20 antiulcer, 21 anxiolytic, 22 diuretic, 23 cardioprotective, 24 antianaphylactic, antiasthmatic, antidepressant, vasodialator and hypotensive effects. 9It is also used in the treatment of oligospermia, aphrodisiac, infertility and benign prostatic hyperplasia.The leaves and roots are used in tuberculosis, cough and against skin infections such as ringworm and wounds. 8The therapeutic potential of this herb is because of the presence of organic acids, flavonoids, triterpenes, steroids, volatiles, amino acids, glucosides, tannins, inorganic ions and lignanoids. 9Thus, L. reticulata is a widely distributed plant with immense therapeutic potential.These properties demand a thorough phytochemical analysis, evaluation and validation of this herbal drug for natural product development.In the current literature available, it is observed that most of the studies are focused on either aerial part or in the whole plant of L. reticulata, there are no reports found on the complete phytochemical analysis of different plant parts (leaf, stem and root) separately of L. reticulata using methanol and ethyl acetate solvents.Besides, there is also lacking reports on the comprehensive phytochemical analysis on tissue culture (in vitro) callus samples generated from this plant.Therefore, this study is focused to evaluate and analyze plant parts and callus extracts of L. reticulata for the presence of phytoconstituents in order to identify and characterize bioactive compounds in the crude extracts prepared in both solvents (methanol and ethyl acetate) for chemical profiling by Gas Chromatography-Mass Spectrometric (GC-MS) technique.The GC-MS is normally used for direct analysis of unknown components existing in traditional medicines and medicinal plants.The results pertaining to GC-MS analysis have led to identification of number of bioactive compounds from L. reticulata sample extracts.They were identified through mass spectrometry attached with GC.This study revealed the accumulation pattern of many known bioactive compounds in different plant parts of L. reticulata and detected many new bioactive compounds which were not earlier reported in this plant.

Collection of Plant Sample
The fresh plant parts of L. reticulata were collected (Priyanka Godara; March 20, 2015) from nearby area of Jaipur, Rajasthan, India.The identity of collected plant specimen was confirmed by depositing the voucher specimen number RUBL211619 in the herbarium of Department of Botany, University of Rajasthan, Jaipur.The plant parts were washed thoroughly with water to remove dust and dried under the shade at room temperature for approximately 15 days till constant dry weight.The dried plant parts were separately ground in liquid nitrogen to obtain the fine powder and kept in an air tight container till further use.

Callus Preparation
The desired amount of callus was produced by sub-culturing of primarily produced callus from leaf explants of L. reticulata on MS media supplemented with standardized amount of growth regulators i.e. 2,4-D (1.25 mg/l) + NAA (0.25 mg/l).

Preparation of Extracts
The dried and fine powdered samples (leaves, roots, stem and callus) of L. reticulata exhaustively extracted with solvents methanol and ethyl acetate.10 g of each powdered plant materials (leaves, stem and roots) and 4 g of callus were packed in thimbles (each in duplicate).Each sample was extracted in methanol and ethyl acetate (100 ml each) separately using Soxhlet apparatus as per standard method.The Soxhlet extracted plant samples were vacuum evaporated using Rotary Vacuum Evaporator (BR Biochem-Scilogex RE100-Pro) to obtain eight dried solid extracts.These extracts were named as Methanolic Leaf Extract (MLE), Methanolic Stem Extract (MSE), Methanolic Root Extract (MRE), Methanolic Callus Extract (MCE), Ethyl Acetate Leaf Extract (EALE), Ethyl Acetate Stem Extract (EASE), Ethyl Acetate Root Extract (EARE) and Ethyl Acetate Callus Extract (EACE).The percent yield of extracts was 9.6% for MLE, 8.7% for MSE, 5.8% for MRE, 4.3% for MCE, 5.1% for EALE, 4.7% for EASE, 2.8% for EARE and 2.6% for EACE.All extracts were stored in vacuum tight container at 4°C in the refrigerator for further use.

GC-MS Analysis
The GC-MS analysis of L. reticulata dried and filtered extracts were carried out on a Thermo GC 1300 and TSQ 8000 Triple Quadrupole GC-MS system installed with auto sampler Al 1310.The program was set using capillary column TG-5MS AMINE (30 mm × 0.25 mm; film thickness 0.25 μm) with initial temperature set to 70°C for 1 min, then gradually increases at 4°C/min up to 270°C with holding time of 1 min.The injector temperature was set at 280°C and the carrier gas used was helium at a flow rate of 1.0 ml/min.GC-MS analysis was conducted using TSQ8000 with transfer line temperature 280°C and ion source temperature 230°C in EI mode.The MS scan parameters included electron impact ionization voltage of 70 eV and a mass range of 50-500 m/z.TSQ 8000 Triple Quadrupole MS detector was used for analysis and data was evaluated using total ion count (TIC) for compound identification and quantification.

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Identification of Bioactive Phytochemicals Phytochemical compounds extracted in different extracts of L. reticulata were identified by comparing the mass spectra of the detected components with the mass spectral data of known components available in the National Institute of Standards and Technology (NIST) library.Compound concentrations were calculated from the GC peak areas of the total ion current (TIC).

Steroids
Steroids were present in different plant part extracts of L. reticulata in abundance and a total of 10 steroidal compounds were detected.Amongst all eight extracts highest amount of total steroidal content was detected in MSE (28.04%) followed by MRE (23.89%),MLE (19.59%),EALE (13.31%),EASE (9.87%) and EARE (6.03%).Steroidal compounds were surprisingly absent in both the callus extracts.Phytosterols were extracted in higher amounts in methanolic extracts.Phytosterols are very well known for their blood cholesterol lowering activity. 25][27][28] Stigmasterol was present in all plant parts but it was found absent in ethyl acetate stem extract and both the callus extracts.It was found in MSE (6.71%), MRE (6.04%), MLE (4.79%), EALE (3.65%) and EARE (0.99%).
Stigmasterol is an unsaturated phytosterol and has various medicinally important properties like cholesterol-lowering, anti-osteoarthritic, 29 thyroid inhibitory, antiperoxidative, hypoglycemic, 30 antihepatotoxic, anti-inflammatory, antinociceptive, antiviral, cancer-preventive activities and sedative effects. 31It is a strong antioxidant and shows antibacterial activity against multidrug resistant mycobacteria. 32β-sitosterol was found in good quantities in methanolic stem (13.40%) and root extracts (9.27%).It was absent in all ethyl acetate extracts.β-sitosterol shows various biological activities like immunomodulatory, antioxidative, hepatoprotective activity 33 and used in the treatment of benign prostatic hyperplasia. 34β-sitosterol shows antiviral effect against Hepatitis B Virus, HIV virus and tobacco mosaic virus (TMV). 33irst time reported from L. reticulata in this study, γ-sitosterol or Clionasterol was found in MSE (12.54%),EALE (6.86%), EASE (6.35%) and EARE (2.59%).γ-sitosterol shows anticancerous, 35 hepatoprotective, anti-hyperglycemic activity and may act as a potential antidiabetic drug. 36,37The Presence of Campesterol is detected in L. reticulata for the first time in this study.It was found in MRE (12.54%),EALE (6.86%), EASE (6.35%) and EARE (2.59%).Campesterol is a cholesterol absorption reducing agent and shows anticancer and antioxidant activities. 38-sitosterol and Campesterol are also used as biomarkers for cancer prevention. 28Cholesterol Myristate or Cholest-5-en-3-ol(3beta)-tetradecanoate is a cholesterol fatty acid ester and detected in ethyl acetate stem extract (0.61%).Cholesterol Myristate is helpful in brain disease therapies, stem-cell transplantation, bone-marrow transplantation, treatment of bone diseases such as osteoporosis and chemotherapy.

CONCLUSION
Leptadenia reticulata (Jeevanti) is a traditional medicinal plant and found to be a rich source of bioactive phytochemicals possessing various biological activities.This study presented a comprehensive analysis of leaf, stem, root and callus extracts in methanol and ethyl acetate.The outcome of GC-MS analysis of this study, presented a detailed comparison of various classes of bioactive phytochemicals identified from different plant parts and callus.A comparative analysis of total 77 different bioactive compounds have been done and further discussed in details on the basis of potential biological role as well as medicinal importance.The present study has first time reported some potential bioactive phytochemicals in L. reticulata that includes γ-sitosterol, Campesterol, Pristane, Hexahydrofarnesol, Stearic acid, Arachidic acid, Coniferyl alcohol, n-Tetracosanol-1, Ascorbic acid 2,6-dihexadecanoate, (2S,3S)-3,7,4'-Trihydroxy-5-methoxy-6-methylflavanone etc.As the medicinal value of similar bioactive components in other plant extracts are already proved, no wonder if these phytochemicals in L. reticulata may also have equal efficacy.It could be concluded that all eight extracts of L. reticulata contain significant abundance of various bioactive compounds from the various categories of bioactive compounds such as steroids, terpenes, fatty acids and their esters, alcoholic and phenolic compounds, hydrocarbons etc.The GC-MS analysis particularly of callus extracts showed high accumulation of fatty acids which was not generally seen in leaves that were used as explant.Further, this study may provide future

Table 1 : Cont'd. Compound type Compound name Molecular formula
ƚ indicates the retention time of the compound present in higher concentration among methanolic and ethyl acetate extracts of leaves, root, stem and callus.
Myristic acid and Stearic acid are saturated fatty acids and were detected in significant amounts only in methanolic leaf, stem and root extracts.Myristic acid or Tetradecanoic acid was found in MSE (2.07%), MRE (1.70%) and MLE (1.36%).Myristic acid shows positive effects on HDL cholesterol and hence improving extracts such