|Ahead of print publication
Plant part substitution in Litsea Chinensis for medicinal use: A comparative phytochemical approach
Bhavana Srivastava1, Vikas Chandra Sharma1, SC Verma2, R Singh3, AD Jadhav1
1 Department of Chemistry, Regional Ayurveda Research Institute for Drug Development, Gwalior, Madhya Pradesh, India
2 Department of Chemistry, Pharmacopoeia Commission for Indian Medicine and Homoeopathy, Ghaziabad, Uttar Pradesh, India
3 Department of Chemistry Central Council for Research in Ayurvedic Sciences, New Delhi, India
Regional Ayurveda Research Institute for Drug Development, Aamkho, Gwalior . 474 009, Madhya Pradesh
Source of Support: None, Conflict of Interest: None
Background: Medasakha (Litsea chinensis) is a medium sized tree, heartwood of which is extensively used in Ayurveda for the treatment of various diseases. Count of this species is declining in the wild and in future this species may be difficult to obtain for use in Ayurveda and other traditional systems of medicine. It is exploited mainly for its medicinal heartwood. Hence the use of alternative parts of this plant in place of its heartwood would be beneficial for its survival. Objective: Present study is carried out on L. chinensis to phytochemically evaluate the possibilities of using its small branches as a substitute to its heartwood. Materials and Methods: Physicochemical parameters and preliminary phytochemical screening were carried out using standard methods. Total phenolic and total flavonoid contents were estimated spectrophotometrically using Folin-ciocalteu and aluminum chloride method, respectively. CAMAG HPTLC system equipped with semi-automatic applicator was used for HPTLC profiling. n-Hexane, ethyl acetate and ethanol extracts of heartwood and small braches were developed in suitable mobile phase using standard procedures and visualized in UV 254, 366 nm and in white light after derivatization with anisaldehyde-sulphuric acid reagent. Results: Phytochemical analysis and HPTLC profile of different extracts of heartwood and small branches showed the presence of almost similar phytochemicals in both the parts of this plant which suggests that small branches may be used in place of heartwood and vice-versa after comparison and confirmation of same for pharmacological activities. Conclusion: This study provides the base for further study to use small branches of L. chinensis as a substitute to its heartwood.
Keywords: HPTLC profile, Litsea chinensis, phytochemical analysis
| Introduction|| |
In Indian systems of medicine most of the medicinal plants are slow-growing trees, bulbous and tuberous plants, with bark, heartwood and underground parts being the parts mainly utilized. Destructive harvesting of these plant parts is leading to the reduction of their natural populations and this is of great concern for resource managers. Forest trees are highly vulnerable to such excessive exploitation and leads to concern regarding the availability of these plants in the future. Therefore, conservation and protection of these plants from extinction has become a matter of urgency. The possible strategies for this may be to establish conservation areas and enforce laws against collecting these plant parts, large-scale cultivation and to encouraging healers to collect and use alternative plant parts such as aerial parts instead of stem bark, heartwood and underground parts. Substitution with the alternative vegetative renewable parts in place of heartwood, bark or underground part of the same plant is likely to be much better in terms of acceptance. A prerequisite for the pursuit of this policy, however, is the evaluation of differences and similarities between various parts of the same plant with respect to chemical composition and pharmacological properties. Possibilities of substitution of heartwood, stem bark and underground parts with aerial parts as a strategy for conservation has been studied by researchers in many plants.,,,,,
Litsea chinensis (Family: Lauraceae) commonly called Medasakha is a useful tree widely used in Ayurveda. As per Ayurvedic literature, wood of this plant is used in śūla (pain), agnimāndya (dyspepsia), śotha (edema), atisāra (diarrhea), raktasrāva (bleeding disorders)and vātavikāra (Rheumatoid arthritis). Aileyaka taila and Vātaghna Lepa are the important formulations of this drug Heartwood is also reported for various pharmacological activities such as antibacterial and aphrodisiac activities and mainly contains alkaloids (laurotetanine, actinodaphine, boldine, norboldine), butenolides, (3R, 4S, 5S)-2-hexadecyl-3-hydroxy-4-methylbutanolide, litsealactone C, litsealactone D, litsealactone G and benzoic acid derivative eusmoside C.
The count of this tree species is declining in the wild mainly because it is exploited for its medicinal heartwood. The plant is reported as red listed plant and considered as critically endangered in Andhra Pradesh, India and as endangered species in Philippines. To face its global demand and scarcity of this medicinal plant, it becomes obligatory to save this plant. In this regard it is requisite to find out a substitute for its heartwood. The present study is an attempt to evaluate the possibilities of using small branches of L. chinensis in place of its heartwood. Standard physicochemical parameters of small branches of L. chinensis have not been prepared yet. So, efforts were also carried out to establish preliminary physicochemical standards of small branches.
| Materials and Methods|| |
Heartwood [Figure 1] and small branches [Figure 2] of L. chinensis were collected from Ghatigaon, Gwalior (M. P), India. Plant material was identified and authenticated by the botanist of the Institute, a voucher specimen was deposited in the Institute.
CAMAG HPTLC system (Muttenz, Switzerland) equipped with semi automatic TLC applicator Linomat IV, twin trough plate development chamber, Win CATS software version 1.4.2 and Hamilton (Reno, Nevada, USA) Syringe (100 μl).
Material and reagents
All chemicals, reagents and solvents used during the experiments were of analytical grade and HPTLC plates were purchased from E. Merck Pvt. Ltd.(Mumbai, India).
Heartwood and small branches were studied for various physicochemical standards such as foreign matter, loss on drying at 105°C, total ash, acid-insoluble ash, alcohol soluble extractive, water soluble extractive and pH of 10% aqueous solution using standard methods.,
Preliminary phytochemical screening
n-Hexane, ethyl acetate and ethanol extract of both heartwood and small branches were screened for the presence of phenols, tannins, carbohydrates, alkaloids, saponins, amino acids, steroids, flavonoids, coumarins, quinone, furanoids and terpenoids by the standard methods of Harborne and Kokate et al.
Estimation of total phenolic and flavonoid content
Five grams of each of the shade-dried plant material was pulverized into a coarse powder and subjected to ethanolic extraction using soxhlet apparatus. Extracts were concentrated to dryness. Dried residues were then dissolved in 100 ml of 95% ethanol. Extracts were used for total phenolic and flavonoid assay.
Total phenolics content was determined by using Folin-ciocalteu assay. An aliquot (1 ml) of extracts or standard solution of gallic acid (20, 40, 60, 80 and 100 μg/ml) was added to a 25 ml volumetric flask, containing 9 ml of distilled water. A reagent blank was prepared using distilled water. One milliliter of Folin-ciocalteu phenol reagent was added to the mixture and shaken. After 5 min, 10 ml of 7% Na2 CO3 solution was added to the mixture. Volume was then made up to the mark. After incubation for 90 min at room temperature, absorbance against reagent blank was determined at 550 nm with an UV/Vis spectrophotometer. Total phenolics content was expressed as μg gallic acid equivalents (GAE).
Total flavonoid content was measured by aluminum chloride colorimetric assay. An aliquot (1 ml) of extracts or standard solutions of quercetin (20, 40, 60, 80 and 100 μg/ml) was added to a 10 ml volumetric flask containing 4 ml of distilled water. To the flask, 0.3 ml of 5% NaNO2 was added and after 5 min, 0.3 ml of 10% AlCl3 was added. After 5 min, 2 ml of 1M NaOH was added and the volume was made up to 10 ml with distilled water. Solution was mixed and absorbance was measured against the blank at 510 nm. Total flavonoid content was expressed as μg quercetin equivalents (QUE).
HPTLC studies were carried out following methods of Sethi, Stahl and Wagner et al. Heartwood and small branches were powdered coarsely. Ten gram of powdered samples of heartwood and small brancheswere accurately weighed and exhaustively extracted by n-hexane, ethyl acetate and ethanol (each 100 ml) separately using soxhlet apparatus. Extracts were filtered and concentrated under reduced pressure and made up to 10 ml in standard flasks separately.
Mobile phase used for developing n-hexane and ethyl acetate extracts of heartwood and small branches was toluene: ethyl acetate (8:2 v/v) and for ethanol extract of heartwood and small branches was chloroform and methanol (in a ratio of 8:2 v/v). Samples were spotted in the form of bands of width 10 mm with a 100 μl Hamilton syringe on aluminum TLC plates pre-coated with Silica gel 60 F254 of 0.2 mm thickness with the help of TLC semi-automatic applicator Linomat IV attached to CAMAG HPTLC system, which was programmed through Win CATS software version 1.4.2. 10 μl of each extracts of heartwood and small brancheswere applied in two tracks as 10 mm bands at a spraying rate of 10 s/μl. Track 1 was heartwood and track 2 was small branches for each of extracts applied. Development of plate up to a migration distance of 80 mm was performed at 27 ± 2°C with mobile phase for each extracts in a CAMAG HPTLC chamber previously saturated for 30 min. After development, the plate was dried at 60°C in an oven for 5 min and visualized under wavelength 254 nm and 366 nm for ultra violet detection. Developed plate was then dipped in anisaldehyde sulphuric acid reagent for derivatization and dried at 105°C in hot air oven till color of band appears and is visualized under white light. Images were captured by keeping plates in photo documentation chamber and Rf values were recorded by Win CATS software.
| Results|| |
Quantitative determination of Physicochemical parameters such as foreign matter, loss on drying at 105° C, ash values, acid insoluble ash, extractive values and pH are given in [Table 1]. Both the parts of L. chinensis were found to possess little moisture and hence can be stored at room temperature without fear of spoilage.
|Table 1: Physicochemical parameters of heartwood and small branches of Litsea chinensis|
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Preliminary phytochemical analysis of different extracts of heartwood and small branches are shown in [Table 2]. Results reveal the presence of similar phytochemicals in heartwood and small branches of n-hexane, ethyl acetate and ethanol extracts.
|Table 2: Phytochemical analysis of extracts of heartwood and small branches of Litsea chinensis|
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Comparative HPTLC profile of n- hexane, ethyl acetate and ethanol extracts of heartwood and small branches of L. chinensis were recorded to reveal the chemical pattern of each extract.
HPTLC profile of n-hexane extract of both heartwood and small branches [Figure 3] and [Table 3] showed no band when visualized under UV at 254 nm. At UV 366 nm heartwood and small branches showed three and two bands but no band found similar. Band at Rf0.60 was found common to both parts but with different color. Visualization under white light after derivatization with anisaldehyde sulphuric acid reagent, heartwood and small branches showed four and six bands, respectively out of which three bands at Rf0.34 (purple), 0.48 (purple), 0.96 (purple) were found similar.
|Figure 3: HPTLC profile of n-hexane extracts of heartwood and small branches of Litsea chinensis (track 1: heartwood, track 2: small branches)|
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HPTLC profile of ethyl acetate extract of heartwood showed no band and small branches [Figure 4] and [Table 4] showed two bands when visualized under UV at 254 nm. At UV 366 nm heartwood and small branches showed two and six bands, respectively out of which one bands at Rf0.63 (blue) was found similar. When visualized under white light after derivatization with anisaldehyde sulphuric acid reagent both heartwood and small branches showed five bands and all were found similar.
|Figure 4: HPTLC profile of ethyl acetate extracts of heartwood and small branches of Litsea chinensis (track 1: heartwood, track 2: small branches)|
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HPTLC profile of ethanol extract of both heartwood and small branches [Figure 5] and [Table 5] showed one band with similar Rf when visualized under UV at 254 nm. At UV 366 nm, both heartwood and small branches showed four bands again with similar Rf. When visualized under white light after derivatization with anisaldehyde sulphuric acid reagent, heartwood and small branches showed four and two bands respectively and both the bands at Rf0.38 (grey), 0.59 (grey) shown in the HPTLC profile of heartwood were also found present in small branches.
|Figure 5: HPTLC profile of ethanol extracts of heartwood and small branches of Litsea chinensis (track 1: heartwood, track 2: small branches)|
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| Discussion|| |
As Ayurveda is growing globally, the number of products emerging in the market, derived from medicinal plants is also increasing. This is resulting in a great demand for raw materials due to which many medicinal plants are facing the threat of becoming extinct or endangered. Harvesting of medicinal plants on such a scale is not sustainable. Therefore, there is an urgent need for a suitable strategy to solve this problem, so that these plants may be available in future for use in Ayurveda and other traditional systems of medicine. It has been observed that in these systems of medicine the most frequently used medicinal plants are slow-growing forest trees, bulbous and tuberous plants with bark, heartwood and underground parts being the parts mainly utilized. Removing bark, heartwood and underground parts of the plant may affect the growth of plants. Only large-scale cultivation cannot solve this problem. So, there is a strong need to look at the challenge from a different perspective. Another approach to deal with the scarcity of medicinal plants would be the use of different parts of the same plant which would satisfy the requirements of sustainable harvesting, yet at the same time provide the healthcare need. At this point, an important question that may arise is whether this approach would be in tune with the basic precepts of Ayurveda. Evidences are available in classical Ayurvedic texts that such steps do indeed get sanction from the tradition. Many references from the classical texts, point to the fact that it is quite justifiable to make use of alternate parts of the same plant when the official part is not available. A prerequisite for this strategy, however, is the evaluation of differences and similarities between various parts of the same plant with respect to chemical composition and pharmacological properties. With this as the background the present study was undertaken on L. chinensis to evaluate the possibilities of using small branches in place of its heartwood which will help sustainable utilization. Data of physicochemical parameters [Table 1] may be useful to pharmaceutical industries for authentication and batch to batch consistency of commercial samples. Almost similar results for qualitative phytochemical analysis of various extracts of heartwood and small branches of L. chinensis [Table 2] indicate the presence of analogous compounds in both the parts of this plant and are of great importance for scientists because healers may substitute the plant parts of the same plant they traditionally use. Quantity of active phytochemicals such as total phenolics and total flavonoids in small branches of L. chinensis [Table 6] indicate that in comparison to heartwood, small branches had high total phenolic and flavonoid contents. This indicates that small branches may possess more active compounds compare to heart wood.
|Table 6: Total phenolic and total flavonoid content of ethanolic extracts of heartwood and small branches of Litsea chinensis|
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In spite of the availability of many sophisticated analytical techniques, thin-layer chromatography still continues to be an important method ideally suited to obtain a characteristic analytical fingerprint of a plant extract, identification of its constituents, determination of impurities and quantification of active substances as it guarantees reproducible results. Comparative evaluation of HPTLC profiles of n- hexane, ethyl acetate and ethanol extracts of heartwood and small branches carried out to reveal the chemical pattern showed many similar bands which again indicated the presence of many similar compounds in heartwood and small branches of L. chinensis. Similarities inboth phytochemical analysis [Table 2] as well as HPTLC profiles of various extracts of heartwood and small branches [Figure 3], [Figure 4], [Figure 5], [Table 3], [Table 4], [Table 5] suggest that small branches may have nearly similar active constituents to that of the heartwood and may be investigated in detail for use as a substitute of heartwood. The above results demonstrate the possibilities of plant part substitution in this plant. However, phytochemical investigations can never substitute pharmacological investigations in determining the therapeutic value of the plant material, so further investigations on comparison and confirmation of the substitutes for pharmacological activities is needed to support the findings. There are many plants for which the official part cannot be harvested in sufficient quantities and therefore, investigations like this may protect more species from extinction, and allow the recovery of threatened medicinal plants. It can provide greater scope for the physicians to utilize raw drugs that are easily available, cost effective and most appropriate for the clinical conditions. We therefore, suggest that every investigation on underground parts or heartwood of medicinal plant may include an investigation on aerial parts of plant also, even though those might not be the parts traditionally used. This study is an initiative to add solution inputs to global concern for the management of traditional medicinal plant resources which has become a matter of urgency today. Results of qualitative evaluation of HPTLC profiles will also be helpful in the identification and quality control of the drug and can provide standard HPTLC profiles with selected solvent system. The HPTLC profiles can also be used as references for the proper identification/authentication of the drug.
| Conclusion|| |
Exploring the possibility of using different parts of the same plant seems to be a relevant approach to tackle the challenge of availability of medicinal plants. In this regard it is necessary to evaluate differences and similarities between various parts of the same plant with respect to chemical composition and pharmacological properties. Similarities in phytochemical analysis and HPTLC profile of various extracts of heartwood and small branches of L. chinensis suggests that small branches of this plant may have almost similar active constituents to that of the heartwood and may be suggested as a substitute of heartwood after comparison and confirmation of same for pharmacological activities. Hence, the study provides the base for further study to recommend small branches of L. chinensis in place of its heartwood which can save this plant from destruction. Investigations like this, may be helpful to form effective strategies towards conservation of bioresources and sustainable bioprospecting specially of medicinal plants.
Authors are thankful to CCRAS, New Delhi for financial assistance and Director General, CCRAS, New Delhi for providing facilities.
Financial support and sponsorship
Central Council for Research in Ayurvedic Sciences, 61-65, Institutional Area, Opp.-D Block, Janakpuri, New Delhi-110058, India.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Zschocke S, Rabe T, Taylor JL, Jäger AK, van Staden J. Plant part substitution – a way to conserve endangered medicinal plants? J Ethnopharmacol 2000;71:281-92.
Sulaiman CT, Balachandran I. Plant part substitution for medicinal use in Aegle marmelos
– A phytochemical approach. J Trop Med Plants 2013;14:19-22.
Srivastava B, Sharma VC, Vashisth E, Singh R, Pandey NK, Jadhav AD. Comparative physicochemical, phytochemical and HPTLC study of stem bark versus small branches of Flacourtia indica.
JSci Innov Res 2015;4:227-31.
Srivastava B, Sharma VC, Sharma H, Pant P, Jadhav AD. Comparative physicochemical, phytochemical and high performance thin layer chromatography evaluation of heart wood and small branches of Aquilaria agallocha
roxb. Int J Ayurveda Pharma Res 2016;4:1-6.
Srivastava B, Sharma VC, Singh R, Pant P, Jadhav AD. Substitution of roots with small branches of Rauwolfia serpentina
for therapeutic uses – A phytochemical approach. Ayushdhara 2015;2:373-8.
Srivastava B, Sharma VC, Singh A, Singh R, Jadhav AD. Evaluation for substitution of heartwood with small branches of Acacia catechu
for therapeutic use – A comparative phytochemical approach. J Pharmacogn Phytochem 2016;5:254-8.
The Ayurvedic Pharmacopoeia of India. Part 1. Vol. 5. New Delhi: Government of India, Ministry of Health and Family Welfare, Department of Indian System of Medicine and Homeopathy; 2008.
Prusti A, Mishra SR, Sahoo S, Mishra SK. Antibacterial activity of some Indian medicinal plants. Ethnobot Leafl 2008;12:227-30.
Ageel AM, Islam MW, Ginawi OT, Al-Yahya MA. Evaluation of the aphrodisiac activity of Litsea
Chinensis (auraceae) and Orchis malculata
(Orchidaceae) extracts in rats. Phytother Res 1994;8:103-5.
Agrawal N, Pareek D, Dobhal S, Sharma MC, Joshi YC, Dobhal MP, et al.
Butanolides from methanolic extract of Litsea glutinosa.
Chem Biodivers 2013;10:394-400.
Reddy KN, Reddy CS. First red list of medicinal plants of Andhra Pradesh, India – Conservation assessment and management planning. Ethnobot Leafl 2008;12:103-8.
Mukherjee PK. Quality Control of Herbal Drugs. New Delhi: Business Horizons Pharmaceutical Publishers; 2008.
Indian Herbal Pharmacopoeia. Indian Drug Manufacturers Association. Revised ed. Mumbai: Indian Herbal Pharmacopoeia; 2002.
Harborne JB. Phytochemical Methods. A Guide to Modern Techniques of Plant Analysis. 3rd
ed. New York: Hall Chapman and Co.; 1998.
Kokate CK, Purohit AP, Gokhale SB. Pharmacognosy. 39th
ed. Pune, India: Nirali Prakashan; 2007.
Singleton VL, Rossi JA Jr. Colorimetry of total phenolics with phosphomolybdic phosphotungstic acid reagents. Am J Enol Viticult 1965;16:144-58.
Zhishen J, Mengcheng T, Jianming W. The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chem 1999;64:555-9.
Sethi PD. High Performance Thin Layer Chromatography Student. 1st
ed., Vol. 10. New Delhi: CBS Publishers and Distributors; 1996.
Stahl I. Thin Layer Chromatography. A Laboratory Hand Book. Berlin: Springer-Verlag; 1969.
Wagner H, Bladt S. Plant Drug Analysis a Thin Layer Chromatography. 2nd
ed. Germany, Atlas: Springer-Verlag; 1984.
Harisastri PV. Ashtanga Hridayam. Varanasi: Chaukhambha Orientalia; 2002. p. 737.
Mitra J. Astanga Sangraha. Varanasi: Chaukhamba Sanskrit Series Office; 2012. p. 582.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]