Impact of seasonal variation on lupeol content in latex of Calotropis gigantea R.Br. and Calotropis procera Ait.
Anagha Ranade1, Rabinarayan Acharya2, Sudipta Roy2, Shivangi Bhardwaj3.
- Regional Ayurved Institute for fundamental research, Pune, Maharashtra, India.
- Dept. of Dravyaguna, IPGT& RA, Gujarat Ayurved University, Jamnagar.
- Pharmaceutical laboratory, IPGT& RA, Gujarat Ayurved University, Jamnagar.
Address for correspondence:
Dr Rabinarayan Acharya, Dept of Dravyaguna, IPGT& RA, Gujarat Ayurved University, Jamnagar, Gujarat 361008.
Ayurveda recommends collection of latex from some plants belonging to Upavisha (semi-poisonous) category in Sharada rutu. The latex from Calotropis gigantea and Calotropis procera (Arka species) finds a significant place, in different therapeutic formulations, with unique mention in management of mukhakaarshnya (hyper pigmentation of face).
Aim: To study the impact of seasonal variation on lupeol content of two species of Arka. Material and methods: The latex was collected aseptically during six seasons from two species. After suitable sample preparation, it was used for HPTLC wherein lupeol was used as standard biomarker. Lupeol is a well known bioactive reported for its anti-inflammatory and anti-melanogenic effect on skin.
Observations and results: The lupeol content of Calotropis gigantea was found to be highest in latex sample collected in August-September (Varsha rutu) whereas it was highest in December-January (Hemanta rutu) in Calotropis procera.
Conclusion: The findings of highest percentage of lupeol in Saumya rutu i.e Hemanta & Varsha rutu thus coincides with the concept of Kala. It thus proves that seasonal variation has an impact on lupeol content of latex.
Keywords: Arka, Calotropis gigantea, Calotropis procera, HPTLC, lupeol, seasonal variation.
In current state of affairs, Ayurveda, is exponentially gaining ground, but faces a major impediment regarding quality assurance and quality control of herbal drugs. Ayurveda recommends many factors, for obtaining good quality crude of herbal origin drugs with potent active principles, namely Desha (habitat), Kala (time) and Guna (properties). WHO, through Good Agriculture and Collection Practices (GACP), also focuses on significance of optimal season or time period for harvesting of medicinal plant yield. Many scientific studies have been carried out to prove the relevance of time factor in deciding the quality of drugs. 
There is a unique description regarding cosmetic application of ksheera (latex) of a poisonous plant (upavisha) especially Arka which botanically corresponds to three varieties of Calotropis. The period of collection of latex is in October and November (Sharada rutu). The physicochemical changes and altered FTIR patterns during six seasons (rutu) in latex of Calotropis gigantea R.Br.(CG) and Calotropis procera Ait.(CP) has been already reported. A thorough review depicted a deficiency in information related to influence of seasons on latex profile using biomarker through HPTLC.
Besides, Calotropis latex has also been reported in cosmetic application pertaining to arrest hyperpigmentation.  Thus, a quantitative analysis of latex of two species (namely Calotropis gigantea and Calotropis procera) in six different seasons was carried out. The lupeol content was considered as a standard biomarker owing to its activity on hyperpigmentation. [10,11] This quantitative analysis of lupeol is an attempt to study the influence of seasonal variation on latex profile.
Material and methods:
Identification and authentication of plant:
The plant Arka with different coloured flowers i.e. purple and white, growing naturally in abundance in the peripheral areas of Gujarat Ayurveda university, Walkeshwari nagari, Sapada road, Jamnagar, Gujarat were identified by local Vaidya and taxonomist. Their respective botanical names i.e. Calotropis gigantea L.RBr. and Calotropis procera Ait. were confirmed by studying the morphological characters, of various parts and comparing them with various characters described in different floras and books. Sample specimens were authenticated by expert of pharmacognosy laboratory of Gujarat Ayurved University, Jamnagar. The herbarium of each sample has been deposited to institute’s pharmacognosy museum which consisted of white variety of Calotropis gigantea Linn. having voucher specimen number (Phm/6147) and purple variety of Calotropis procera Ait. having voucher specimen number (Phm/6149).
The fresh crude latex of both C.gigantea (CG) and C.procera (CP) were collected by three tender and fresh parts of the twig in clean glass vials in morning time regularly in all the six rutu i.e Vasanta (March-April), Grishma (May-June), Varsha (July- August), Sharada (September- October), Hemanta (November- December) and Shishira (January- February)[table 1]. The time for collection of latex in all rutu was kept constant (within 7 am to 9 am) and in second week of each rutu to avoid sandhikala. One ml of each samples of latex were mixed in equal quantity of 5% alcoholic KOH and subjected to heat.  Later, the methods has been modified in order to obtain unsaponified layer by adding the mixture with 3 ml of Chloroform (analytical grade of Merck) and separated by separating funnel and stored in airtight glass vials. Lupeol (purity 95% by GC), the reference standard was procured from Natural remedies Pvt. Limited; Bangalore and used for quantification.
Table 1: The six rutu and their corresponding Hindu lunar months along with Gregorian month
|Sr.no.||Rutu||Season||Hindu Lunar month||Gregorian month|
|1.||Vasanta||Spring||Chaitra & Vaishakha||March- April|
|2.||Grishma||Summer||Jyeshtha & Ashadha||May –July|
|3.||Varsha||Rainy season||Shravana & Bhadrapada||August-September|
|4.||Sharada||Autumn||Ashvina & Kartika||October- November|
|5.||Hemanta||Pre-winter||Margashira & Paushya||December-January|
|6.||Shishira||Winter||Magha & Phalguna||February –March|
Method of preparation of Standard solution lupeol:
A stock solution of 1mg/ml of lupeol was prepared in HPLC grade methanol and used for establishing its linearity at 3 concentrations of lupeol viz. 2.5 µg, 5 µg and 7.5µg as depicted in figure below-
The HPTLC quantification was done using Camag Linomat V as the application mode consisting of development chamber of Camag twin trough make. The stationary phase consisted of precoated silica gel GF254 plates. The chamber saturation time was kept for 30 mins where as the development time was 30 mins. The development distance was fixed at 8 cms. The scanner used was of Camag scanner III make. The detection system comprised of Deuterium lamp, Tungsten lamp with WinCat software as the Data analysis system. The mobile phase used was toluene: ethyl acetate (7:3)  .Visualization of the spots was done by dipping the plate in Vanillin Sulphuric acid and scanning done at 524 nm.
After the scanning was done in to the Camag Scanner III, densitograms obtained were studied for the area versus concentration at maximum obtained Rf value.
Observations and results:
Table 2: Concentration of lupeol in six rutu:
|CG Śiśira||0.83||5903.7||4.209 µg|
|CP Śiśira||0.80||3260.9||2.325 µg|
|CG Vasanta||0.80||3037.7||2.165 µg|
|CP Vasanta||0.79||1266.4||0.902 µg|
|CP Grīṣma||0.74||1667.2||1.188 µg|
|CG Varṣā||0.73||6851.3||4.88 µg|
|CP Varṣā||0.74||6405||4.5 µg|
|CG Śarada||0.74||6683.8||4.76 µg|
|CP Śarada||0.75||3719.7||2.65 µg|
|CG Hemanta||0.75||908.7||0.64 µg|
|CP Hemanta||0.68||7071.3||5.041 µg|
The highest quantity of lupeol was present in C.procera in Hemanta and in C.gigantea in Varsha rutu (Table 2); (Plate 1; Fig1-15).
The study was planned to assess the effect of time factor i.e six seasons in influencing lupeol content in crude latex of Calotropis gigantea L. and Calotropis procera Ait. Lupeol is a pentacyclic triterpene which has a wide therapeutic usage. The anti-inflammatory, anti-oxidant and skin conditioning activity has added to its therapeutic significance.  Lupeol is also reported to support epidermal regeneration, with potential applications in formulations that target skin texture and integrity.  As it has been reported in Calotropis species and possesses cosmetic value as mentioned above, lupeol was chosen as a marker for seasonal variation in this study.
The highest quantity of lupeol was present in C.procera in Hemanta and in C.gigantea in Varsha rutu (Table 2); (Plate 1; Fig1-15). Lupeol being a phytosterol in nature, can be correlated to Saumya nature. The findings of highest percentage Saumya rutu  i.e Hemanta & Varsha rutu thus coincides with the concept of Kala.
The latex is thus a rich source of lupeol and if collected in the appropriate season can prove beneficial in sunscreen formulations. But, species wise difference observed in lupeol quantity may be due to varied plant physiology. Thus, the time factor plays a significant role in plant physiology.
The highest quantity of lupeol is present in Hemanta rutu in C.procera while in Varsha rutu C.gigantea. Further studies for assessing the pharmacological activities of lupeol in various seasons can lead to definite conclusions for standardising the specific collection period of Calotropis latex.
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Conflicts of Interest
Authors have no competing interests.
- Acharya JT., Editor. Charaka Samhita. commentary of Chakrapani. Kalpasthana. 1/7, 1st Ed., Chaukhambha Prakashana, Varanasi, Reprint 2011; 652.
- Anonymous. WHO guidelines on good agricultural and collection practices (GACP) for medicinal plants (PDF). World health organisation. Geneva: 2003. 11.
- Acharya RN, Ranade AV. Fostering the concept of Kala-The Relevance of time factor in augmenting crude drug quality. In: Dhiman KS. Padhi MM.Mangal AK. Srikanth N. Editors. Recent trends in Good agricultural and Collection practices for medicinal plants. First edition. New Delhi: CCRAS. Ministry of Ayush. pp 323-344.
- Shastri L. Yogaratnakar. First edition. Chaukhambha Prakashana, Part II. Varanasi, Reprint 2010; 281.
- Ranade Anagha V, AcharyaRabinarayan. Arka and its botanical equivalents: A critical analysis. Ayurpharm Int J Ayur Alli Sci. 2015;4(3); p 60-68.
- Acharya JT., Editor. Charaka Samhita. commentary of Chakrapani. Kalpasthana. 1/10, 1st Ed., Chaukhambha Prakashana, Varanasi, Reprint 2011; 653.
- Ranade AV, Acharya RN, Shukla V and Roy S. Exposition of Role of Diurnal and Seasonal Variation on Latex of Calotropis procera Ait. and Calotropis gigantea L.R.BR. European Journal of Medicinal plants. 2017;19(1):1-7.(Published on 25th May 2017.)
- Ranade AV, Acharya R, Shukla V, Roy S, Maji J. Monitoring Of Seasonal Variation In Latex Of Calotropis Procera AIT. And Calotropis Gigantea L.R.Br Using Ftir Spectroscopy. J Res Educ Indian Med – ISSN 0970-7700. (2017), 23(1-2): 59-74. (April –June 2017)
- Shastri L. Yogaratnakar. First edition. Chaukhambha Prakashana, Part II. Varanasi, Reprint 2010; 281.
- Azizuddin, A. Majeed Khan & M. Iqbal Choudhary. Tyrosinase inhibitory potential of natural products isolated from various medicinal plants, Natural Product Research; 2011, 25:7, 750-753.
- Singh M, Suman S, Shukla Y. New enlightenment of skin cancer chemoprevention through phytochemicals: in vitro and in vivo studies and the underlying mechanisms. BioMed research international. 2014; pp 1-18.
- Anonymous. The Ayurvedic Pharmacopoeia of India e-book Part 1. Volume 6. New Delhi: Govt. of India: Ministry of Health and Family Welfare, Department of Indian Systems of Medicine and Homoepathy; 2001. Appendix 3.3.7.
- Leela V, Saraswathy A. Quantification of pharmacologically active markers Gallic Acid. Quercetin and Lupeol from Acacia Leucophloea Willd flowers by HPTLC Method. IRJP.2013; 4(2):146-150.
- Fernandez MA, et al New insights into the mechanism of action of the anti-inflammatory triterpene lupeol. J. Pharm Pharmacol. 2001; 53(11):1533-9.
- Nikiema JB et al Effects of anti-inflammatory triterpenes isolated from Leptadenia hastata latex on keratinocyte proliferation. Phytother Res. 2001. 15(2):131-4.
- Sharma PV. Editor Sushruta Samhita with Nibandhasangraha of Dalhana. Sutrasthana 36.verse 5. 9th edition. Chaukhambha orientalia, Varanasi, 2007; 159.