PHARMACOGNOSTIC AND HPTLC COMPARISON OF KERALA MARKET SAMPLES OF BHARANGI (CHERUTHEK) WITH GENUINE SOURCE PLANT ROTHECA SERRATA (L.) STEANE & MABB

PHARMACOGNOSTIC AND HPTLC COMPARISON OF KERALA MARKET SAMPLES OF BHARANGI (CHERUTHEK) WITH GENUINE SOURCE PLANT ROTHECA SERRATA (L.) STEANE & MABB

A. Navami *, P. Vivek and C. M. Harinarayanan

Department of Dravyaguna Vijnana, VPSV Ayurveda College, Kottakkal, Malappuram, Kerala, India.

ABSTRACT: Background: Bhāraṅgī, herbal drug used in Ayurveda as Kapha-Vata samaka and indicated in Swasa, Kasa, Vrana, Shotha etc. It is an important ingredient in many of the preparations like Kantakaryavaleha, Kanakasava, Dasamularista, Lodhrasavam, Sudarsanasavam. As per API the accepted source of Bhāraṅgī is Rotheca serrata (L.) Steane & Mabb belongs to family Lamiaceae. There were ambient studies in which it was found that roots of Bhāraṅgī had been adulterated with stem of the same in the market. But there were no published works indicating the presence of adulterants in the Kerala market, hence this study was proposed to assess the genuineness of raw drug available in the name of Cheruthek (Bhāraṅgī) in different Kerala markets with the root of genuine source plant R. serrata. Materials and Methods: Fresh root of Bhāraṅgī (Rotheca serrata (L.) Steane & Mabb) are collected from the natural habitat. The market samples of Cheruthek were collected from six different regions in Kerala. All samples were screened for pharmacognostic parameters- macroscopic, microscopic, histo-chemical and powder microscopic characterization along with HPTLC profile and compared with the genuine drug. Result & Conclusion: After the pharmacognostic comparison and HPTLC profiling of six market samples of the drug Cheruthek collected from selected districts of Kerala, the Kozhikode sample had similar characters of the R. serrata; Thiruvananthapuram sample was having characters of the plant Premna herbaceae Roxb. All other samples were adulterated with various plant parts.

Keywords: Bhāraṅgī, (Rotheca serrata (L.) Steane & Mabb, Pharmacognosy, Market study, Adulteration, Ayurveda

INTRODUCTION: Ayurveda is increasingly gaining recognition as one of the medical sciences worldwide in the last few decades. Plant materials were used throughout the world as home remedies, over-the-counter drug products and raw materials for the pharmaceutical industry, and represent a substantial proportion of the global drug market. Commercialization in the medical system leads to substituting or adulterating herbal drugs to withstand scarcity.

Using commercially available inferior-quality drugs had compromised the therapeutic efficacy of Ayurveda medicine. Hence, the quality standardization of drugs had got greater importance in Ayurveda. As per CCRAS, a market sample survey has been found to be one of the priority areas for drug research ¹.

Bhāraṅgī, a potent Ayurvedic drug used in respiratory disorders, which acts as Kapha Vata Samaka. In classics the roots were considered as the part used along with root bark which is used in many of the Ayurvedic formulations like Bharangyadi kashaya, Bharangyadi churna, Kantakaryavaleha, Bhāraṅgī Guda, Kanakasava, Lodhrasava etc. As per Ayurvedic Pharmacopeia of India and Quality Standards of Indian Medicinal Plants, states that Rotheca serrata (L.) Steane & Mabb belongs to the family Lamiaceae is the botanical source of Bhāraṅgī and the official part used is dried roots ^{2, 3}. Due to increased demand and over-exploitation, there is a significant reduction in the availability of genuine drug; hence chances for adulteration with other spurious, inferior or useless parts of same or different plants is very high. Some previous studies found that the roots of Bhāraṅgī are adulterated with the stem, in many markets. From a previous study conducted on the market samples of Bhāraṅgī, it was found that all the samples were adulterated and none of the samples were genuine (Singh et. al. 2015) ⁴. The most of the samples obtained in South Indian are observed to be the roots and root nodules of Pygmaeopremna herbacea (syn. Premna herbacea) (Vasudevan et. al. 1971) ⁵. Though Brihattrayis and Nighantukara have described only one variety of Bhāraṅgī, we come across two types of Bhāraṅgī in Vaidyaka Sabda Sindhu, the white flowered and blue flowered. This description has become a source for controversy. The latter authors clarified that the white-flowered variety is identified as Clerodendrum indicum (L.) Kuntze and the blue flowered one as Rotheca serrata (L.) Steane & Mabb ^{6, 7}. There were no published works are available about the genuinity of Bhāraṅgī in Kerala market samples. Hence in this study, mainly aimed to compare the different market samples of Bhāraṅgī (Cheruthek) by Pharmacognostic and High-performance thin layer chromatography (HPTLC) profiling with the root of genuine source plant Rotheca serrata (L.) Steane & Mabb.

MATERIALS AND METHODS: The root and stem of genuine sample of Bhāraṅgī (Cheruthek) were collected from Herbal Garden of Vaidyaratnam P. S. Varrier Ayurveda College, Kottakkal and Vrindavanodyanam, Thanal Mathrusadanam, Mayanoor, Thrissur. The samples were authenticated and specimens were deposited at the Herbarium of Center for Medicinal Plants Research (CMPR), Arya Vaidya Sala, Kottakkal, Kerala. (Herbarium Voucher No: 10047). The different market sample of Bhāraṅgī (Cheruthek) were procured from six different markets of Kerala. (Thiruvananthapuram, Idukki, Palakkad, Kozhikode, Wayanad and Kasargod) The genuine sample of Rotheca serrata (L.) Steane & Mabbwere compared with the market samples, on basis of pharmacognostic parameters along with HPTLC profile.

Samples were named as follows:

Samples 1: Roots and stem of Rotheca serrata (L.) Steane & Mabb

Sample 2: Sample collected from Thiruvananthapuram market.

Sample 3: Sample collected from Idukki market.

Sample 4: Sample collected from Palakkad market.

Sample 5: Sample collected from Kozhikode market.

Sample 6: Sample collected from Wayanad market.

Sample 7: Sample collected from Kasargod market.

Sample 8: Root of Clerodendrum indicum.

Methodology: External characters were studied and documented per standard API protocol. The materials for anatomical study were processed as per the standard protocol. The powder analysis and maceration study were done. Observations were done under Leica DM 1000 LED microscope and photographs were taken using Leica DFC 295 in-built camera. For HPTLC profiling of the water extracts of the samples were done under the solvent system - Ethyl acetate: methanol: water (9:1:1). Observed under UV light at 254, 366nm and after derivatization with ANS reagent, recorded the R_f value. Densitometric scanning of plates was done using Camag TLC scanner 3 at 254, 366 and 550nm.

RESULTS: The genuine sample of Bhāraṅgī and its six market samples were compared by its pharmacognostic parameters using macroscopic, microscopic, histochemical, powder microscopic characters along with HPTLC profile.

Macroscopic Comparison: Organoleptic characters of the Samples of Bhāraṅgī were shown in Table 1 and Fig. 1. Samples showed almost identical characteristics such as colour, fractures, external markings and cut surfaces. The main differences observed between the samples were in thickness. The sample 1; root and stem had extremely different texture compared to market samples. Sample 6 showed extremely different macroscopy when compared to others.

TABLE 1: MACROSCOPIC COMPARISON OF THE SAMPLES OF BHĀRAṅGĪ

Microscopic Comparison:

Microscopy of Sample 1 (Root and Stem):

Root: TS of the root was almost circular in outline with outer layers of cork ruptured at many places followed by a wide cortex, phloem and well- developed xylem region. Detailed TS showed a well-developed cork region with two distinct zones - outer zone consisting of up to 15 layers of comparatively thick walled rectangular to tangentially elongated cells which often peeled off at many places. Inner zone composed of more than 15 layers of tangentially elongated radially arranged cells. Cortex was composed of 10 - 15 layers of oval or elongated loosely arranged parenchymatous cells with inter-cellular spaces. Simple and compound starch grains measuring upto 10-15μm in diameter are scattered throughout the cortical region along with acicular and rod-shaped calcium oxalate crystals.

Solitary and groups of fibers and stone cells present throughout the cortical region. Phloem is comparatively narrow composed of compactly arranged compressed cells often alternating with medullary ray cells at many places. Simple starch grains and acicular and rod-shaped crystals of calcium oxalate were present at some places.

Centre portion occupied by xylem elements such as vessels, fibers, parenchyma and medullary rays. Xylem vessels were round to oval in shape, 30-100μm diameter usually in solitary and rarely grouped. Medullary rays 2-3 seriated composed of radially elongated pitted parenchymatous cells often filled with starch grains and acicular crystals of calcium oxalate. Starch grains also observed in xylem elements including parenchyma cells and fibers. The presence of growth rings in mature roots was observed in Table 2 and Fig. 2.

Stem: TS of the stem was almost circular in outline with narrow cork layers which were ruptured at many places followed by wide cortex, narrow phloem and well-developed xylem region. Detailed TS showed narrow cork region with 3-5 layers of rectangular to tangentially elongated cells. Cortex were composed of 10-15 layers of oval to elongated loosely arranged parenchymatous cells with intercellular spaces. Pericyclic fibers seen in cortical region in discontinuous patches alternating with solitary or group of stone cells. Starch grains of diameter 10-15μm and acicular crystals present in some cortical cells. Phloem narrow composed of compactly arranged compressed cells often alternating with medullary ray cells. Xylem region composed of vessels, fibers and medullary rays. Xylem vessels round to oval in shape, 40-150μm diameter usually solitary and rarely in grouped and scattered al-throughout the xylem region.

Tyloses present. Xylem fibers thick walled and possess major part of xylem region. Medullary ray uni-bi seriated composed of radially elongated pitted parenchyma cells. Pith is comparatively large and occupies the central region, composed of parenchymatous cells filled with starch grains and acicular and rod-shaped crystals of calcium oxalate at some regions Table 2 and Fig. 2.

Microscopic Comparison with Market Samples: The TS of the Thiruvananthapuram sample and Idukki sample had dissimilar microscopic characteristics with that of genuine sample. The presence of orange-brown deposition at the inner layers of cork cells were evidently seen in the Thiruvananthapuram and Idukki samples.

The presence of stone cells was very few in the cortical region in the Thiruvananthapuram sample and Idukki sample. While its abundant in case of genuine sample. The presence of cortical fibers were found in the root samples of Thiruvananthapuram sample and Idukki sample. Starch grains and acicular crystals were present comparatively less with genuine sample. Presence of oil globules in case of the Thiruvananthapuram sample and Idukki sample. Palakkad and Kozhikode samples had similar microscopic characteristics to genuine samples.

The presence of stone cells and fibers was even in both samples, starch grains and acicular crystals of calcium oxalate. Wayanad and Kasargod samples did not resemble any of the microscopic characters of the genuine sample. The Wayanad sample prominently showed the vascular region which is dissimilar with that of genuine. The Kasargod sample showed different TS of root and stem pieces. It possesses uni-bi seriated medullary rays. Presence oil globules were seen in both samples Table 2, Fig. 3.

TABLE 2: MICROSCOPIC COMPARISON OF THE SAMPLES OF BHĀRANGĪ

HPTLC Profile: HPTLC chromatograms and densitometric profiles of 8 samples at 254nm, 366nm and 550 nm were recorded. Fig. 4 the Rf values of separated compounds of each sample were noted. Table 3 At 254 nm, sample 5 has maximum number of peaks (10 peaks), sample 8 with 8 peaks, and sample 4 with 7 peaks. Rf values of many peaks of these samples were similar indicating same chemical constituents. Rotheca serrata (L.) Steane & Mabb. root sample (sample 1R) had only seven peaks and its stem (sample 1S) with 8 peaks. Sample 6 shows very less peaks. Peak intensities were varied in case sample 2 and sample 3. Sample 5 showed some unique peaks that were observed in the genuine stem and root.

TABLE 3: Rf VALUE OF SAMPLES

DISCUSSION: Quality and Safety are fundamental principles in the provision of herbal medicines for health care. Scientific studies estimate herbal product adulteration as 40-60% in India, the most of Indian herbal medicinal products are essentially mixed with one or a few other herbs that could lessen the therapeutic activity of the main ingredients ⁸. Only a few studies are available on quality standards of some market samples.

A study conducted in South Indian Market samples of Bhāraṅgī, it was noted that the roots of C. serratum was the accepted species for the drug. As per previous study other species coming as Bhāraṅgī were Gardenia latifolia, G. resenifera, G. turgid, Premna intergrifolia and Picrasma quassioides. However, it has been observed that in South Indian Pharmacies, the root and root nodules of Pygmacopremna herbacea were used as Bhāraṅgī in their preparations (K. Vasudevan Nair et.al 1982)⁵. In a study conducted on the four Indian market samples of Bhāraṅgī, it was found that all samples were adulterated and none of the market sample was found as genuine drug Clerodendrum serratum ^{3, 4, 9}. Another study showed that the roots of Bhāraṅgī were adulterated with the stem of Clerodendrum serratum, mostly in South Indian markets ^{10, 11}. Eventually there were no particular studies conducted on Bhāraṅgī in Kerala markets. This study thus aimed to conclude the exact identity of the market sample of Bhāraṅgī with special regards to Kerala markets. After undergoing macroscopic examination, morphologically sample 4, 5 & 7 had stout, cylindrical roots similar to genuine root; even then, sample 4 & 7 had a mixture of stem and roots. Sample 2 and 3 possess stoloniferous roots that don’t match the original sample ie. Sample 1 which was collected from Mayyanoor, Thrissur.

There were differences in the dimensions and thickness. Sample 5 had similar characters as that of sample 1. Sample 6 was having extremely different morphological characters when compared with the genuine root. Hence, the chances of genuine roots of Bhāraṅgī coming in the market is comparatively less based on macroscopical observations. Sample 2 & 3 had similar morphological characteristics of that of roots of Premna herbacea Roxb. In a previous study also stated that sample collected from Southern Kerala can of roots of Premna herbacea Roxb ⁴. In-order to compare the microscopic character of Thiruvananthapuram sample, previous study in which the pharmacognostic evaluation of roots of Pygmaeopremna herbacea (syn. Premna herbacea.) discusses the macroscopic, microscopic characteristics along with HPTLC profile of the Premna herbacea Roxbwere noted (Rastogi. S et. al. 2005) ¹². None of the samples had similarities with the sample 8 C. indicum ⁷.

TS of genuine root of R. serrata was compared with that of 6 market samples. There was extreme difference in case of the Thiruvananthapuram sample (sample 2) and Idukki sample (sample 3) with that of genuine one. Presence of cortical fibers in patches rather than bundle of stone cells. Absence of starch in root samples and the presence of oil globules in both the samples. There was presence of orange-brown colour depositions in the inner cork layers unlike original sample. The Palakkad sample (Sample 4) and Kozhikode sample (sample 5) possess similar microscopic characteristics of that of genuine sample. Palakkad sample possess the stem pieces which did not have the microscopic characteristics of the stem of R. serrata. Presence of oil globules were noted in both the samples. Starch grains were present but less abundant as compared to genuine sample. Wayanad sample (sample 6) and Kasargod sample (Sample7) had no significant similarity with genuine sample. Wayanad sample had well developed xylem resembling a heart wood. Phloem was wide, compressed with brown depositions, fibers are scattered. In Kasargod sample, one of the root pieces showed similar characters of genuine one other sample are different hence it’s a mixture of plant parts. None of the samples had similarities with the sample 8 C. indicum.

The HPTLC comparison of water extracts of different samples were carried out. HPTLC chromatograms and densitometric profiles of nine samples at 254nm, 366nm, and 550nm were recorded. The Rf values of the separated compounds of each sample were noted. At 254nm, root of R. serrata had only seven peaks and the stem had eight peaks. The peak intensities were different for each sample, the peak intensities of sample 5 had similarity with that of genuine sample i.e. sample 1. Sample 4 also show some similar peaks but less. Similar Rf values uniformly present in all the samples studied indicate the presence of similar phyto-constituents. Similarly, at 366nm, root of R. serrata had only seven peaks and the stem had nine peaks. Sample 5 shows more similar bands with the sample 1 when compared with others, indicating the presence of a greater number of chemical constituents than the original one. There were no previously published comparable works to conclude these differences in the number of peaks of different samples. After analyzing the result in Table 4, it was clear that the raw drugs available in the market in the name of Cheruthek (Bharangi) were not derived from a single botanical source, revealing the practice of adulteration and substitution in the raw drug industry.

TABLE 4: INTERPRETATION OF MARKET SAMPLES

CONCLUSION: The samples of the drug Bhāraṅgī (Cheruthek) collected from 6 different markets from different districts of Kerala were pharmacognostically different, except the sample collected from Kozhikode. The organoleptic and microscopic evaluation of the market samples revealed that only Kozhikode sample had similar characteristics with that of the genuine sample of Bhāraṅgī- Rotheca serrata (L.) Steane & Mabb. Even though the genuine part used was root, Kozhikode sample possess both the stem and root. HPTLC profiling also revealed that similar peaks with different band thickness may be due to differences in maturity, soil, climate, and time of collection of drugs. From the previous reports, the Thiruvananthapuram sample can be the roots of Premna herbacea one of the adulterants of the drug Bharangi. The Idukki sample too contains roots of Premna herbacea along with other mixtures. The Palakkad sample contains the roots and stems of R. serrata and other mixtures. The Wayanad and Kasargod samples were completely different and had no similarity with the source plants R. serrata & C. indicum. Hence, it was clear that none of the market samples were only root but a mixture of root and stem.

FIG. 1: SAMPLES OF BHARANGI ALONG WITH SAMPLES FROM DIFFERENT MARKETS IN KERALA. A-B. Genuine sample bharngi; a. root of rotheca. b. stem of Rotheca serrata; c. root of Clerodendrum indicum; d. Thiruvananthapuram market sample; e. Idukki market sample; f. Palakkad market sample g. Kozhikod market sample, h. Wayanad maekwt sample; i. Kasargod market samole.

FIG. 2: MICROSCOPIC CHARACTERS OF THE ROOT AND STEM OF R. SERRATA. A-G. TS OF ROOT. A. TS of root portion enlarged; b. TS portion inlarge showing outer portion; c. TS cork region enlarged; d. ts showing stelar region; e. and f. enlarged view of cortical region; g. xylum region portion enlarged; h-n. TS of stem; h. t sod stem portion enlarged; i. TS portion; k. pericycle fibers l. xylume vessels m. enlarged cortical region n. parenchyma cells of pith ck, cork; clcr, crystals of calcium oxalte; ct, cortex; f, fiber; mr. modularly rays; ph phloem; sg starch grains; stc, stone cells; x, xylem; xf, xylem fibers; xv, xylem vessels

FIG. 3: A-Z: MICROSCOPY OF MARKET SAMPLE OF BHARANGI. A-C. TS of thiruvanthapuram market sample; D-G. TS of Idukki market sample; H-L. TS of palakkad markets saple; M-Q. TS of Kozhikode market sample; R-W. TS of wayanad sample; X-Z. TS of kasargod market sample; 1-5 microscopy of Clerodendrum indicum.

FIG. 4: HPTLC COMPARISON OF GENUINE AND MARKET SAMPLES OF BHARANGI. A-c. TLC profile of sample at 254nm, 366nm after derivatisation; d-f; denitometeric profile at 254nm 366nm and after derivatisation. 1. Rotheca serrata rot, 2.Rotheca serrata stem, 3. Thiruvananthapuram sample, 4. Idikki sample, 5. Palakkad sample, 6. Kozhikode sample, 7. Wayanad sample, 8. Kasargod sample, 9. Root of Clerodendrum indium.

ACKNOWLEDGEMENT: The authors are grateful to Dr. N. Manoj Kumar, Prof. and Head; Dr. Madhu K P Associate Prof., Dr. Vidya Unnikrishnan, & Dr. Jyolsna G Krishna, Assistant Prof.; Department of Dravyaguna Vijnana, VPSV Ayurveda College, Kottakkal; Mr. Deepak M, Scientist, Phytochemistry Division & Ms. Haritha, Technical Assistant, Pharmacognosy Division, CMPR, AVS, Kottakkal, Kerala for their help and support during the study.

Financial Support and Sponsorship: Nil

CONFLICTS OF INTEREST: There was no conflict of interest.

REFERENCES:

1. Sharma PC, Yelne MB and Dennis TJ: Database on Medicinal Plants Used in Ayurveda. Vol New Delhi. Central Council for Research in Ayurveda & Siddha 73-75.
2. Anonymous: The Ayurvedic Pharmacopoeia of India. 1st ed. Part 1, Volume III. Delhi: the Controller of Publications Civil Lines, Delhi 2001; 25-26.
3. D CD, R KY and B VP: Abhava pratinidhi dravya: A Comparative Phytochemical Study of Bharangi and Kantakari. IJAM [Internet]. 2021 Sep. 29 [cited 2022 Sep. 30]; 12(3): 588-92. Available from: https://ijam.co.in/index.php/ijam/article/view/2070
4. Singh RK, Singh A, Nathani S and Murthy AR: Cardinal Identification Features of Bharangi and its Market Samples. Int J of Ayur and Herb Med 2015; 5(2): 1689-94.
5. Vasudevan Nair K, Yogasimhan SN, Keshavamurthy KR and Mart Z: Studies onsome South Indian market samples of Ayurvedic drugs-I. Ancient Sci Life 1971; 2: 71-78.
6. Shastri JLN: Textbook of Dravyaguna Vigyanana. Reprint 2010. Vol. 2.Varanasi: Chaukhamba Orientalia, Varanasi. 2010; 422.
7. Lahari Sidde, Malathi S, Swethalatha S and Rajani K: A Breif Review on Clerodendrum indicum. International Journal of Indigenous Herbs & drugs 2018; 3(5): 1-4.
8. Shanmughanandhan D, Raghupathy S, Newmaster SG, Mohanasundaram S and Sathishkumar R: Estimating herbal product authentication and adulteration in India using vouchered DNA based biological reference material library. Drug Saf 2016; 39: 1211-27.
9. Gadade Jayashree and Patil Swaroopa A: Rotheca serrata: an Overview. Int J of Life Scie Special 2018; 10: 185-190.
10. Narayanan N, Thirugnanasambantham P, Viswananthan S, Sukumar E and Jeyaraman P: Pharmacognostical Studies on the Roots of Clerodendrum serratum. Pharmaceutical Biology 2002; 40(5): 362-368, DOI:10.1076/phbi.40.5.362.8454.
11. D CD, R KY and B VP: Abhava pratinidhi dravya: A Comparative Phytochemical Study of Bharangi and Kantakari. IJAM [Internet]. 2021 Sep. 29 [cited 2022 Sep. 30]; 12(3):588-92. Available from: https://ijam.co.in/index.php/ijam/article/view/2070
12. Rastogi Subha, Madan Mohan, Kumar K, Rawat AKS and Pushpangadan P: Pharmacognostical Evaluation of Roots of Pygmaeopremna herbacea (Roxb.) Mold. Naturals Product Sciences 2005; 11(2); 58-62.
    

    ---

    How to cite this article:

    Navami A, Vivek P and Harinarayanan CM: Pharmacognostic and HPTLC comparison of Kerala market samples of Bharangi (Cheruthek) with genuine source plant Rotheca serrata (L.) steane & MABB. Int J Pharm Sci & Res 2023; 14(12): 5676-86. doi: 10.13040/IJPSR.0975-8232.14(12).5676-86.

    ---

    All © 2023 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.