QUALITY ANALYSIS OF MARKET SAMPLES OF ŌRILA COLLECTED FROM DIFFERENT MARKETS OF KERALA USING PHARMACOGNOSTIC TOOLS

QUALITY ANALYSIS OF MARKET SAMPLES OF ŌRILA COLLECTED FROM DIFFERENT MARKETS OF KERALA USING PHARMACOGNOSTIC TOOLS

A. A. Athira *, K. P. Madhu and C. M. Harinarayanan

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

ABSTRACT: Ōrila is a well-known drug used in many Ayurvedic recipes in kerala. In most texts Ōrilais seen paired with Mūvilaand together they fare as components of the Daśamula. From very old Kerala commentaries of ancient Ayurvedic texts, Ōrila and Mūvila are seen as Malayalam translations of Pṛśniparṇī and Śālaparṇī respectively. In Kerala Pṛśniparṇīis often identified as Ōrila and its source here is Pleurolobus gangeticus (L) J. St.-Hil. However, in North India and in API, source plant of Pṛśniparṇīis Uraria picta (Jacq.) Desv. ex DC. Both belongs to the family Fabaceae where former is a stout herb or under shrub and the other is a small branched, perpetual herb. In the current globalized world where market have expanded, there are unconfirmed reports that other drug sources like Uraria pictaetc have begun appearing in Kerala herbal drug market in the name of Ōrila. So, decided to investigate the status of samples of Ōrila available in the Kerala herbal drug market and to check the quality and authenticity of the samples using pharmacognostic tools. As per microscopic studies, samples from Trivandrum, Kozhikode and Wayanad contains P. gangeticus roots and stems along with other similar looking plants. Idukki, Palakkad and Kasargod samples were mixtures of some similar looking species of Desmodium. Malappuram contains roots and stems of Mūvila (Pseudarthria viscida). On HPTLC analysis similar bands matching with P. gangeticus roots and stems were observed in Trivandrum, Kozhikode and Wayanad samples.

Keywords: Pṛśniparṇī, Market sample study, Pleurolobus gangeticus, Desmodium gangeticum, Śālaparṇī, Pharmacognosy

INTRODUCTION: Ayurveda, the science of life, is one of the oldest traditional systems of medicine. The knowledge is gained from Vedas, mainly based on Atharvaveda. Later Samhitas, Sangraha grant has, Nighanṭus, commentaries with translations came and modern era texts compilation came into being. Ayurvedic literature is vast as said above & it got translated to different languages, so we can see the name of a single drug source in Sanskrit and other local or vernacular names.

Language became a problem and it is evident when it came to commentaries. As India is a country having vast area and diversity due to its geoclimatic condition, a single drug can have multiples sources in different regions. Therefore, there is a chance of mixing up of drugs.

In last 2- 3 decades, due to the decreased availability, enlarged market, faster raw-drug logistics and establishment of new pharmaceutical companies demand for a particular raw drug can be met from different regions and the essential nature of local raw-drug market is being transformed. Therefore, in this scenario, there is a high relevance for a study on the market availability of drugs. In such a background, this study focuses on the drug Ōrila, a well-known drug used in many Ayurvedic recipes and often seen paired with Mūvila and together they fare as components of the famous drug group Daśamūla. Very old Kerala commentaries of ancient Ayurvedic texts such as Aṣṭāṅgahṛdaya both Ōrila and Mūvila are seen as Malayalam translations of Śālaparṇī and Pṛśniparṇī. In Kerala Pṛśniparṇī is often identified as Ōrila and its source here is Pleurolobus gangeticus (L) J. St.-Hil ¹. However, in North India and in Ayurveda Pharmacopoeia ofIndia, the source plant of Pṛśniparṇīis Uraria picta (Jacq.) Desv. ex DC²botanically very different from Pleurolobus gangeticus (L) J. St.-Hil. The larger question about the exact identity of Śālaparṇī   and Pṛśniparṇī is there due to the above said problems which is not settled yet. However, this study is not taking these problems into consideration. As far as this study concerned, the single problem is Ōrila andits natural source in south India which is Pleurolobus gangeticus (L) J.St.-Hil. And whether the market reflects this identified source and if it does not, then attempt to identify what more is being sourced as Ōrila. This study is important when Ōrilais alone in a formulation i.e., not paired with Mūvila. In Malayalam texts like sahasrayoga and Malayalam commentaries of classical texts such as Aṣtāngahṛdaya, commentators translate the term Sthirā as Ōrila in yogas such as that of Rasonadi kaṣāyam. That may cause problem due to the change in the formulation and effect, by the usage of Uraria picta (Jacq.) Desv. ex DC instead of Pleurolobus gangeticus (L) J. St.-Hil.

In the Study Substitutes of Śālapaṛnī (Desmodium gangeticum): A Conceptual Review concluded that in the absence of Desmodium gangeticum, other five species (D. podocarpum, D. repandum, D. triffolium, D. diffusum, and D. lexiflorum) of Genus Desmodium can be considered as substitutes on the base of Karma, Pharmacognostical morphology and some of the common chemical constituents of them ³. And also there present study on an ethnobotanical survey on adulterants of medicinal plants used by traditional practitioners of Palakkad district, Kerala, India showed that adulteration had become a popular practice in the herbal industry & Desmodium gangeticum DC is one of the commonly adulterated drugs ⁴. Therefore, to address a relevant problem, i.e., the possibility of adulteration of Ōrila, the drug which fares in many formulations can cause huge impact in the health of society, this study is being taken up. The main issues facing by thedrug sector is substitution, adulteration ⁵ etc can bring down the genuineness of Ayurveda drug field. Due to the informal activity of complex levels of suppliers, from the primary level of collectors to the specific dealers with the big manufacturing companies, the market is not controlled by any specific regulatory body in the state. In the current globalized world where market have expanded, herbals drug sources are no longer geographically limited. As a result, there are unconfirmed reports that other drug sources such as Uraria picta (Jacq.) Desv. ex DC etc. have begun appearing in the Kerala herbal drug market in the name of Ōrila and today there is condition where manufacturers or doctors are forced to buy whatever present in the market even without knowing original or not as they are not collecting it directly from authorized collectors. Sandigdha dravy as is a term used for medicinal plants having controversial botanicals as sources. So, we decided to check whether the drug present in the different markets is the same or whether any problem of raw material supply and quality currently exits by doing the comparison of its Pharmacognostic properties and HPTLC study of the market samples with the both source plants of Pṛśniparṇī and Śālaparṇī   as per API.

MATERIALS AND METHODS: Crude market samples were collected from 7 various markets and standard samples were collected from their natural sources.

The samples were named as: -

Sample No. 1: Pleurolobus gangeticus (L) J.St.-Hil Root (P.g- Rt) and Stem (P.g- St) from herbal garden of CMPR Kottakkal.

Sample No. 2: Collected from Thiruvananthapuram Market Sample (T).

Sample No. 3: Collected from Idukki Market Sample (I).

Sample No. 4: Collected Palakkad Market Sample (P).

Sample No. 5: Collected from Malappuram Market Sample (M).

Sample No. 6: Collected from Kozhikode Market Sample (KZ).

Sample No. 7: Collected from Wayanad Market Sample (W).

Sample No. 8: Collected from Kasaragod Market Sample (KS).

Sample No. 9: Aerial parts of Uraria picta (Jacq.) Desv.ex DC. (U. picta) from the garden of J. S Ayurveda Mahavidyalaya, Nadiyad Genuine samples were collected, authenticated and Herbarium specimens were deposited in ‘CMPR Herbaria’ (Pleurolobus gangeticus (L) J.St.-Hil. - CMPR HERBERIA 12373, Uraria picta (Jacq.) Desv. ex DC- CMPR HERBARIA 12868). Samples were washed under running tap water, air dried under shade, coarsely powdered and were kept in airtight containers for further use. Macroscopic and microscopic characters of the market samples along with HPTLC we reassessed as a part of this study. It was conducted at Pharmacognosy and Phytochemical lab, Dept of Dravyaguna Vijnana, V.P.S.V. Ayurveda College, Kottakkal and Center for Medicinal Plants Research, Arya Vaidya Sala, Kottakkal during the period of April, May and June 2022.

Macroscopic Study: Morphological characters of the genuine samples of Ōrila and its 7 market samples were studied and documented as per API. Characters such as shape, size, colour, surface, odour, and taste were noted and compared with the genuine samples.

Microscopic Study: Thin transverse sections of the root and stem of each sample were taken by hand and using microtome. The microscopical evaluation of sections were carried out to confirm the structural details of the materials. For effective results various reagents or stains were used to distinguish cellular structures. Histochemical tests were also done by applying the stains in required quantities. The compound microscope used for microscopic study was Trilocular Leica DM 1000 LED attached with 'Leica DFC 295' digital camera application software 'LAS version V3’ were used for viewing and transferring microscopic images.

Powder Microscopy: The cell structure and inclusions in the powder were examined by powdering the material with mortar and pestle. Then it was stained by using appropriate stains and mounted in 40% glycerine. It was then observed under microscope with attached camera for transferring microphotographs to the computer connected to it.

Maceration Method: The Maceration of samples were done by standard maceration techniques (Jeffrey's method). Maceration was done by treating small pieces of plant material in a test tube with maceration fluid (10% nitric acid and 10% chromic acid) for 1-2 hrs at 30-40° C. when the material became softened and cells got separated all the maceration fluid was drained out and the material was washed for several times. Then it was stained it with aqueous safranin, mounted in glycerine and observed under the microscope with attached camera for transferring microphotographs to the computer connected to it. Characteristics of lignified cells, xylem elements such as fibres and vessels were noted.

Phytochemical Study: Phytochemical comparison of the two source plants and seven market sample of Ōrila by thin layer chromatography (TLC). For the purpose, parts of selected plants were collected, and dried in the shade. The dried materials were powdered and subjected to extraction. For the extraction, 5 gm each of the plant materials were taken in round bottomed flask and refluxed with 50 ml of distilled water at continuously for 2 hours. The extracts were filtered, concentrated, and made up to 10 ml in a standard flask. Samples were applied on the plate using CAMAG automatic TLC sampler 4 attached to CAMAG HPTLC system. Applied 5 µl of test solution and (1 to 3 μl) of standard solution separately on a precoated silica gel 60 F25 TLC plate (E. Merck) of uniform thickness of 0.2 mm plate in the form of bands with width 8 mm using Hamilton syringe (100μl). The plate being developed in the solvent system in a twin trough chamber to 9 cm. The Stationary phase was Aluminium backed pre-coated Merck silica gel plate 60 F plate (10x10 cm). and the Solvent system used was Toluene: Ethyl acetate: formic acid (7:3: 0.1). The Visualization done by observing the plate under UV light at 254, 366 nm and after derivatisation with ANS reagent, recorded the R-value and colour of the resolved bands and the Densitometric scanning of the plates was done by using CAMAG TLC scanner 3 at 254, 366 and 550 nm.

RESULT AND DISCUSSION: The genuine samples of Ōrila and its seven market samples are compared by its pharmacognostic analysis using macroscopic, microscopic, histochemical, powder microscopic characters along with HPTLC parameters.

Macroscopic and Microscopic Analysis:

Macroscopic Comparison (Fig. 1 & Fig. 2, Table 1):

Macroscopic Characteristics of Pleurolobus gangeticus (L) J.St.-Hil. (Fig. 1):

Root: Drug consists of dried, cylindrical, branching tortuous, lateral pieces of root, pieces of 5to 10 cm in length and 5 to 7 mm in diameter; pieces of tap root being thicker, few and poorly developed, surface is somewhat smooth, light yellowish, shows at places thin wiry rootlets or the scars left by them, transversely running lenticels and bacterial nodules of various sizes, bark is thin easily peel able from the central solid core of wood; fracture, outer short, inner fibrous. Taste is sweet and mucilaginous; odour not characteristic.

FIG. 1: HABIT AND MARCOSCOPY OF THE SOURCE PLANTS OF ŌRILA. A.-F. Pleurolobus gangeticus LG.-J. Uraria picta Jacq

FIG. 2:  SAMPLES OF ŌRILA COLLECTED FROM DIFFERENT MARKETS OF KERALA. A. Thiruvananthapuram Market Sample; B. Idukki Market Sample; C. Palakkad Market Sample, D. Malappuram Market Sample, E. Kozhikode Market Sample; F Wayyanad Market Sample; G. Kasargod Market Sample

TABLE 1: MACROSCOPIC COMPARISON OF SOURCE PLANTS AND SEVEN MARKET SAMPLES OF ŌRILA

*P.g – Pleurolobus gangeticus, T- Thiruvananthapuram, I- Idukki, P- Palakkad, M- Malappuram, KZ-Kozhikode, W- Wayanad, K- Kasaragod, U. picta – Uraria picta.

Stem: Slender, up to 1.0 cm in diameter, branched, somewhat angular, clothed with appressed greyish hairs, external surface brown, internal pale yellow; fracture, short; taste, slightly bitter ⁶.

Macroscopic Characteristics of Uraria picta (Jacq.) Desv.ex DC. (Fig.1):

Root: Occur in pieces of varying size, thickness of 1 to 2 cm, gradually tapering, tough, woody, cylindrical; externally light yellow to buff, internally pale yellow; surface bearing fine longitudinal striations; fracture, splintery or fibrous; taste, slightly acrid.

Stem: About 10-15 cm long, 0.2 - 0.6cm in diameter, in cut pieces; cylindrical, branched, pubescent, light yellow to brown external surface, transversely cut and smoothened surface, longitudinal wrinkles in mature stem, leaf scar present at nodes, fracture fibrous.

Leaves: Imparipinnate, up to 10 cm or more long, up to 2 cm wide, linear- oblong, acute, glabrous above, finely reticulately veined, leaf in upper part 5-7 and in lower part 3-5leaf blotched with white colour.

Macroscopic Comparison with Market Samples (Fig. 2): Mostly mixture of dried sample of Ōrila are available in the market. In the 4 market samples, i.e., from Malappuram, Kozhikode, Wayanad, Kasaragod we collected very small pieces were found.

Macroscopic features are not sufficient for confirming the identity of samples. During our study we got mixtures of samples from almost all the markets except Idukki, Palakkad. Out of which some are finely chopped so that macroscopic features are not clearly distinguishable.

All the samples were mixtures of both roots and stems. In Trivandrum, Idukki, Palakkad samples leaves also were present. The details of the macroscopic comparison are given in the Table 1.

Microscopic Comparison (Fig. 3 – Fig. 11, Table No. 2): Microscopic Characteristics of Pleurolobus gangeticus (L) J.St.-Hil. (Fig. 3 & Fig. 4):

Root: Diagrammatic TS of the root circular to oval in outline with outer layer ruptured at manyplaces, shows cork, cortex, phloem, and xylem. Major portion of the section occupied by wood region and medullary ray cells very prominent.

FIG. 3: MICROSCOPY OF PLEUROLOBUS GANGETICUS L. ROOT. A. TS OF ROOT SHOWING ENTIRE VIEW, B. TS PORTION ENLARGED; C & E. TS OUTER REGION ENLARGED, D. XYLEM REGION ENLARGED. F. TS CORTICAL PORTION ENLARGED; H. ENLARGED VIEW OF XYLEM VESSELS AND FIBRES; G. & L. TEST FOR LIGNIFIED CELLS; 1. TEST FOR TANNIN DEPOSITION; J. & K. TEST FOR STARCH GRAINS; M. PRESENCE OF CRYSTALS CF, CORTICAL FIBRES, CK, CORK, CR, CRYSTALS OF CALCIUM OXALATE; ET, CORTEX, LG, LIGNIN; MR, MEDULLARY RAYS; OG, OIL GLOBULE, PH, PHLOEM, SG. STARCH GRAIN; IN, TANNIN CELL, V, VESSEL, X, XYLEM, XF, XYLEM FIBRES

FIG. 4: MICROSCOPY OF PLEUROLOBUS GANGETICUS L. STEM. A. TS OF SHOWING ENTIRE VIEW, B. TS PORTION ENLARGED; C. TS OUTER REGION ENLARGED, D. PITH REGION ENLARGED, E. TS OUTER SHOWING TRICHOME; F. TS CORTICAL PORTION ENLARGED; G. & 1. ENLARGED VIEW OF XYLEM VESSELS AND FIBRES; H. PRESENCE OF CRYSTALS, J. TS PHLOEM PORTION ENLARGED, K. TEST FOR TANNIN DEPOSITION; L.. TEST FOR STARCH GRAINS, M. TEST FOR OIL, N. TEST FOR LIGNIFIED CELLS, CK, CORK: COL, COLLENCHYMA, CR, CRYSTALS OF CALCIUM OXALATE; ET, CORTEX, EPI, EPIDERMIS, F, FIBRES, LIG, LIGNIN, MR, MEDULLARY RAYS, OG, OIL GLOBULE; PERF, PERICYCLIC FIBRE, PH, PHLOEM, PHF, PHLOEM FIBRE, SG STARCH GRAIN, TN, TANNIN CELL, X, XYLEM, XF, XYLEM FIBRES, V. VESSEL

FIG. 5: MICROSCOPY OF ŌRILA MARKET SAMPLE COLLECTED FROM TRIVANDRAM. A.-D. TS OF ROOT SAMPLE 1; E-H. TS OF ROOT SAMPLE 2, 1.-1. TS OF STEM SAMPLE 1: M-P TS OF STEM SAMPLE 2. CK, CORK: COL, COLLENCHYMA, CR, CRYSTALS OF CALCIUM OXALATE; ET, CORTEX, EPI, EPIDERMIS, F, FIBRES, LIG, LIGNIN, MR, MEDULLARY RAYS, OG, OIL GLOBULE; PERF, PERICYCLIC FIBRE, PH, PHLOEM, PHF, PHLOEM FIBRE, SG STARCH GRAIN, TN, TANNIN CELL, X, XYLEM, XF, XYLEM FIBRES, V. VESSEL

FIG. 6: MICROSCOPY OF ŌRILA MARKET SAMPLE COLLECTED FROM IDUKKI AND PALAKKAD. A.-D. TS OF IDUKKI ROOT SAMPLE, E.-11. TS OF IDUKKI STEM SAMPLE, 1-1. TS OF PALAKKAD ROOT SAMPLE, M. -PTS OF PALAKKAD STEM SAMPLE.CK, CORK: COL, COLLENCHYMA, CR, CRYSTALS OF CALCIUM OXALATE; ET, CORTEX, EPI, EPIDERMIS, F, FIBRES, LIG, LIGNIN, MR, MEDULLARY RAYS, OG, OIL GLOBULE; PERF, PERICYCLIC FIBRE, PH, PHLOEM, PHF, PHLOEM FIBRE, SG STARCH GRAIN, TN, TANNIN CELL, X, XYLEM, XF, XYLEM FIBRES, V. VESSEL

FIG. 7: MICROSCOPY OF ŌRILAMARKET SAMPLE COLLECTED FROM MALAPPURAM. A.-F. TS OF ROOT SAMPLE; G-JTS OF STEM SAMPLE 1; L.-O. TS OF STEM SAMPLE.CK, CORK: COL, COLLENCHYMA, CR, CRYSTALS OF CALCIUM OXALATE; ET, CORTEX, EPI, EPIDERMIS, F, FIBRES, LIG, LIGNIN, MR, MEDULLARY RAYS, OG, OIL GLOBULE; PERF, PERICYCLIC FIBRE, PH, PHLOEM, PHF, PHLOEM FIBRE, SG STARCH GRAIN, TN, TANNIN CELL, X, XYLEM, XF, XYLEM FIBRES, V. VESSEL

FIG. 8: MICROSCOPY OF ŌRILA MARKET SAMPLE COLLECTED FROM KASARGODE. A.-G. TS OF ROOT SAMPLE, H.-K. TS OF STEM SAMPLE 1; L.-O. TS OF STEM SAMPLE 2.CK, CORK: COL, COLLENCHYMA, CR, CRYSTALS OF CALCIUM OXALATE; ET, CORTEX, EPI, EPIDERMIS, F, FIBRES, LIG, LIGNIN, MR, MEDULLARY RAYS, OG, OIL GLOBULE; PERF, PERICYCLIC FIBRE, PH, PHLOEM, PHF, PHLOEM FIBRE, SG STARCH GRAIN, TN, TANNIN CELL, X, XYLEM, XF, XYLEM FIBRES, V. VESSEL

FIG. 9: MICROSCOPY OF ŌRILAMARKET SAMPLE COLLECTED FROM WAYANAD. A. D. TS OF ROOT SAMPLE 1: E.-H. TS OF ROOT SAMPLE 2, 1.-L. TS OF STEM SAMPLE 1; M.-P. TS OF STEM SAMPLE.CK, CORK: COL, COLLENCHYMA, CR, CRYSTALS OF CALCIUM OXALATE; ET, CORTEX, EPI, EPIDERMIS, F, FIBRES, LIG, LIGNIN, MR, MEDULLARY RAYS, OG, OIL GLOBULE; PERF, PERICYCLIC FIBRE, PH, PHLOEM, PHF, PHLOEM FIBRE, SG STARCH GRAIN, TN, TANNIN CELL, X, XYLEM, XF, XYLEM FIBRES, V. VESSEL

FIG. 10: MICROSCOPY OF ŌRILA MARKET SAMPLE COLLECTED FROM WAYANAD. A. D. TS OF ROOT SAMPLE 1: E.-H. TS OF ROOT SAMPLE 2, 1.-L. TS OF STEM SAMPLE 1; M.-P. TS OF STEM SAMPLE.CK, CORK: COL, COLLENCHYMA, CR, CRYSTALS OF CALCIUM OXALATE; ET, CORTEX, EPI, EPIDERMIS, F, FIBRES, LIG, LIGNIN, MR, MEDULLARY RAYS, OG, OIL GLOBULE; PERF, PERICYCLIC FIBRE, PH, PHLOEM, PHF, PHLOEM FIBRE, SG STARCH GRAIN, TN, TANNIN CELL, X, XYLEM, XF, XYLEM FIBRES, V. VESSEL

FIG. 11: MICROSCOPY OF ŌRILA MARKET SAMPLE COLLECTED FROM KASARGODE. A.-G. TS OF ROOT SAMPLE, H.-K. TS OF STEM SAMPLE 1; L.-O. TS OF STEM SAMPLE 2.CK, CORK: COL, COLLENCHYMA, CR, CRYSTALS OF CALCIUM OXALATE; ET, CORTEX, EPI, EPIDERMIS, F, FIBRES, LIG, LIGNIN, MR, MEDULLARY RAYS, OG, OIL GLOBULE; PERF, PERICYCLIC FIBRE, PH, PHLOEM, PHF, PHLOEM FIBRE, SG STARCH GRAIN, TN, TANNIN CELL, X, XYLEM, XF, XYLEM FIBRES, V. VESSEL

 TABLE 2: MICROSCOPIC COMPARISON OF THE SOURCE PLANTS AND MARKET SAMPLES OF ¬ŌRILA

⁸* P. gangeticus – Pleurolobus gangeticus, U.picta – Uraria picta.

Detailed TS shows outer cork region composed of up to 15 layers of tangentially elongated, rectangular cells arranged in radial rows; outer layers ruptured at some places. Some of the cells of outer layer show reddish brown deposition. Cork is followed by a cortical region made up of up to 15 layers of oval to elongated, parenchymatous cells.

Plenty of fibres in solitary and in groups, few small sized starch grains and prismatic crystals of calcium oxalate are scattered throughout the cortical region. Some of the cells are filled with reddish brown depositions. Phloem is seen as small patches above the xylem composed of closely packed parenchymatous cells often alternating with radiallyelongated ray cells. Plenty of prismatic and rod-shaped crystals are scattered throughout the parenchymatous cells of the section. Lignified fibres in single and in groups scattered in the phloem region. Xylem is a wide zone composed of tracheids, fibres, vessels, xylemparenchyma and medullary ray cells. Major portion of the xylem occupied by fibres and lignified parenchyma cells. Xylem fibres found as radial patches alternating with parenchyma cells. Xylem vessels round to oval, having diameter 20 - 60 μm, solitary orin groups, arranged radially at many places throughout the xylem. Tyloses and reddish-brown deposition found in some vessels especially at the centre. Medullary ray uni tomultiseriate composed of radially elongated pitted parenchymatous cells. Ray cells are extended towards the cortical region at many places and shows the presence of prismatic crystals of calcium oxalate and small sized starch grains

Stem: Diagrammatic TS of the stem circular in outline with outer layer ruptured at many places, shows cork, cortex, phloem and xylem. Major portion of the section occupied by wood region. Detailed TS shows outer cork region composed of up to 7 layers of tangentially elongated cells; outer layers ruptured at some places. In young stem single layered epidermis of small, oval parenchyma cells covered with thick brownish cuticle and interrupted at places by multicellular trichomes, a hypodermis consisting of 3 or 4 layers of ovalcollenchyma cells; Cork is followed by a cortical region made up of up to 11 layers ofoval to elongated parenchymatous cells. Pericycle region represented by discontinuous ring of fibre patches. Few small sized starch grains and prismatic crystals of calcium oxalate are scattered throughout the cortical region especially in ray cells. Phloem region composed of up to 10 layers of closely packed parenchymatous cells alternating withradially elongated ray cells. Lignified phloem fibres and plenty of prismatic crystals are scattered throughout this region. Xylem is a wide region composed of tracheids, fibres, vessels, xylem parenchyma and medullary ray cells. Major portion of the xylem occupied by fibres and vessels. Xylem vessels round to oval, having diameter 10 - 50 μm occurring more as solitary or in groups, scattered throughout the region. Medullary ray mostlyuniseriate, rarely biseriate composed of radially elongated pitted parenchymatous cells extend towards the phloem region filled with starch grains. Pith is made up of thin-walled large polygonal shaped parenchymatous cells. Rods shaped crystals present in pith cells

Microscopic Characteristics of Uraria picta (Jacq.) Desv.ex DC (Fig. 5):

Root: Diagrammatic TS of the stem circular in outline with outer layer ruptured at manyplaces, shows cork, cortex, phloem and xylem. Shows 5 or 6 layers of thin-walled, tabular, regularly arranged cork cells; cortex composed of 4 to 6 layers of oval, tangentially arranged, thin-walled, parenchymatous cells, a few fibres present singly or in groups. Phloem parenchyma composed of rounded to somewhat oval cells, larger onwards periphery: fibres thick walled. lignified with narrow lumen and tapering phloem rays 1 to 5 cells wide. Xylem composed of vessels, tracheids, fibres, crystal fibres and parenchyma traversed by xylem rays; vessel very few, mostly confined to inner and outer part of xylem, xylem parenchyma mostly rectangular with simple pits; xylem ray cells is diametric showing simple pits, starch grains simple, round to oval, measuring 6 to 17 u in diameter, distributed throughout parenchymatous cells of secondary cortex, phloem and xylem; prismatic crystals of calcium oxalate present in crystal fibres, as well as in many parenchymatous cells of secondary cortex, phloem and ray cells.

Stem: Diagrammatic TS of the stem circular in outline with outer layer ruptured at many places, shows cork, cortex, phloem and xylem. Major portion of the section occupied by pith region.

Detailed TS shows outer cork region composed of 2-3 layers of tangentially elongated cells ruptured at places, a hypodermis consisting of 2 or 3 layers of collenchyma cells. Below Cork is followed by a cortical region made up of up to that ³ -4 layers of oval to elongated thickened parenchyma cells followed by thin walled loosely arranged parenchyma cells with intercellular spaces. Patches of pericyclic fibre and few prismatic crystals present in that region. Wood area is seen as bundle. Wide phloem region composed of 12 - 14 closely packed parenchymatous cells alternating with radiallyelongated ray cells. Lignified phloem fibres scattered and reddish-brown deposition found. Xylem is a wide region composed of tracheids, fibres, vessels, xylem parenchyma and medullary ray cells. Major portion of the xylem occupied by fibres and parenchyma cells. Xylem vessels round to oval in shape, having diameter um occurring more as solitary or in groups, arranged radially throughout the xylem. Medullary ray mostlyuniseriate and multiseriate composed of radially elongated pitted parenchymatous cells extend towards the cortex region. Wide pith is made up of layer large pitted parenchymatous cells, thick-walled inner layer and thin-walled outer layer with rodsshaped crystals present.

Microscopic Comparison with market Samples (Fig. 6 to Fig. 11): Majority of the root samples showed the presence of reddish- brown deposition in cortical cells. In Malappuram sample very thick deposits were observed. Almost all the stem samples except some of the samples of Trivandrum and Kozhikode and Wayanad showed the presence of reddish-brown deposition. In Malappuram sample there were plenty of content cells in large clusters like that of Pseudarthria viscid and so, Malappuram sample may be the roots and stems of Pseudarthria viscida. Almost all samples showed presence of reddish-brown contents, plenty of vessels and phloem fibres which are not matching with the characters of U. picta mentioned in API. So, can conclude that Kerala market samples do not have the presence of north Indian sample U. picta. Some of the roots selected from Trivandrum samples, root sample of Kozhikode and selected root samples of Wayanad showed the presence of scattered cortical fibre, starch grains and prismatic crystals. Some of the stem sample of Trivandrum and Kasargod showed the presence of rosette crystals of calcium oxalate in cortical cells.

Root of P. gangeticus was with xylem fibres resembling that of phloem which are arranged in radial patches alternating with xylem parenchyma. Xylem area of U. picta were found with round to oval vessels with uni to multiseriate medullary rays containing starch grains. Also, wide pith with large pitted parenchymatous cells containing rod-shaped crystals were present in it. Same arrangement was observed in some of the roots from Trivandrum, Kozhikode and Wayanad. The details of the microscopic comparison are given in the Table 2.

Histo-chemical Comparison:

Histochemical Characteristics of Pleurolobus gangeticus (L) J.St.-Hil:

Root: Small sized round starch grains were present in the parenchyma cells of cortex, medullary ray and in the xylem region. Pericyclic fibres, phloem fibres, xylem elementsand medullary rays lignified. Tannin deposition present in the cork and phloem rayregion. Presence of oil globules not observed.

Stem: Small sized round starch grains were present in the parenchyma cells of cortex and medium sized ones in the xylem region. Pericyclic fibres, phloem fibres, xylem elements and medullary rays lignified. Tannin deposition present in the cork and phloemray region. Also, small oil globules present in the xylem region.

Histochemical Characteristics of Uraria picta (Jacq.) Desv.ex DC

Root: Starch grains in the xylem ray. Prismatic crystals in parenchyma cells of cortex,ray cells, xylem, and phloem. Lignin content present in the xylem elements, pericyclicfibres and in the phloem fibre region.

Stem: Medium sized round starch grains were present in the xylem region. Lignin content present in the xylem elements, pericyclic fibres and in the phloem fibre region. Tannin present in the cork to phloem region. Plenty of small oil globules present in the inner xylem region, phloem region and in the cortex region.

Histochemical Comparison with Market Samples: Histochemical tests of Pleurolobus root and showed small sized round starch grains in parenchyma cells of cortex, medullary ray and in the xylem region where in Uraria picta starch grains were present in xylem region only. Pericyclic fibres, phloem fibres, xylem elements and medullary rays lignified. Tannin deposition present in the cork and phloem rayregion. Small oil globules in xylem of Pleurolobus where plenty of them were found in inner xylem region, phloem region and in cortex of Uraria picta.

In all market samples, histochemical tests for tannin showed deposition in cortex and cork region. Lignification was present in a xylem and cortical fibre region, pericycle fibre, phloem fibre region of all samples. In all samples, starch grains were present in roots and stems with varying areas, sizes, and numbers in the xylem and in cortex region except in the Malappuram. The details of the histochemical comparison are given in the Table 2.

Powder Microscopy (Fig. 12 & Fig. 13):

Powder microscopic Characteristics Pleurolobus gangeticus (L) J.St.-Hil. (Fig. 12):

Root: Surface view of cork cells; fragments of cortical parenchyma cells with and without reddish brown content; prismatic crystals of calcium oxalate; bundle of vessels and fibres; groups of vessels and fibres; pitted parenchyma cells with starch grains measuring 7-25μ in diameter³; red coloured pigmented particle; pitted vessels etc observed in powder.

Stem: Shows surface view of cork cells; prismatic crystals and rod shaped of calcium oxalate; parenchyma cells; fragments of fibres; trichomes; xylem vessels; cut group of xylem vessels and tracheids; fragments of longitudinally cut crystal fibres; parenchyma cells and fibres filled with starch grains; bordered pitted vessel.; pitted parenchyma cells.

FIG. 12: POWDER MICROSCOPY OF PLEUROLOBUS GANGETICUS L. A.- H. CHARACTERS OF ROOT; I. - P. CHARACTERS OF STEM. A. CORK CELLS IN SECTIONAL VIEW; B. CORK CELLS IN SURFACE VIEW; C. PITTED PARENCHYMA CELLS OF MEDULLARY RAY; D. PRISMATIC AND ROD-SHAPED CRYSTALS OF CALCIUM OX STARCH GRAINS. E. FRAGMENTS AND FIBRES AND VESSELS; F. & G. FRAGMENTS OF FIBRES; H. FRAGMANTS OF VESSELS AND FIBRES; I. CORK CELLS IN SECTIONAL VIEW; J. CORK CELLS IN SURFACE VIEW; K. PRISMA ROD SHAPED CRYSTALS OF CALCIUM OXALATE; L. FRAGMENTS OF VESSELS; M. PITTED PARENCHYMA CELLS OF MEDULLARY RAYS; N. FIBRES WITH POINTED ENDS; O. FIBRES WITH STARCH GRAINS; P. FRAGMENTS OF FIBRES AND VESSELS.

FIG. 13: POWDER MICROSCOPIC CHARACTERISTIC OF MARKET SAMPLES; A. - I. CHARACTERS OF ROOT; J.- P. CHARACTERS OF STEM. A. CORK CELLS IN SECTIONAL VIEW; B. CORK CELLS IN SURFACE VIEW AND UNDERLYING CORTICAL CELLS WITH RED CONTENT; C. CORTICAL CELLS IN SECTIONAL VIEW WITH CONTENTS; D. REDDISH BRWON CONTENT; E. PITTED PARENCHYMA CELLS; F. PARENCHYMA CELLS WITH CONTENTS; G. CALCIUM OXALATE CRYSTALS AND PIGMENT CELLS; H. FRAGMENTS OF FIBRES AND VESSELS; I. FRAGMENTS OF FIBRES; J. CORK CELLS IN SURFACE VIEW; K. CORK CELLS IN SECTIONAL VIEW; L. PITTED PARENCHYMA CELLS; M. PARENCHYMA CELLS FROM PITH; N. CORK CELLS IN SECTIONAL VIEW; O. FIBRES AND PITTED VESSELS; P. PITTED PARENCHYMA CELLS.

Powder Microscopic Characteristics of Uraria picta (Jacq.) Desv.ex DC:

Root: Greenish-yellow; shows simple pitted vessels; fragments of fibres, tracheids, parenchyma cells; pieces of hairs; palisade cells; a few prismatic crystals of calciumoxalate; epidermal cells wavy walled in surface view showing paracytic stomata and starch grains simple, round to oval, measuring 6 to 17 u in dia.

Stem: Shows fragments of surface view of cork cells; prismatic crystals and rod shaped of calcium oxalate; parenchyma cells; xylem fibres and phloem fibres; trichomes; pittedxylem vessels; cut group of xylem vessels and tracheids; fragments of longitudinally cutcrystal fibres; parenchyma cells and fibres filled with starch grains; bordered pitted vessel.; pitted parenchyma cells in pith; spiral vessel.

Powder Microscopic Comparison with Market Samples (Fig. 13): Powder microscopy of market sample showed the presence of Rod-shaped crystals of calcium oxalate were found only in Pleurolobus and Trivandrum sample only. Simple and compound starch grains in clusters and solitary, Prismatic crystals, bundle of vessels and fibres, parenchyma cells with starch grains pitted vessel, fragments of surface view of cork cells, parenchyma cells, xylemfibres, trichomes, fragments of xylem vessels fibres, fragments of longitudinally cutcrystal fibres with or without reddish content, pitted parenchyma cells and fibres, pitted vessel, bordered pitted vessel where in the market sample reddish brown deposition were more.

HPTLC Comparison (Fig. 14 & Fig. 15, Table 3): The Rf value of the genuine source plants and the market samples of Ōrila were calculated and colour of the bands were also noted. The Rf values at 254nm, 366nm & Rf value visualized after derivatisation are shown in the Table 3.

FIG. 14: HPTLC COMPARISON OF THE HOT WATER EXTRACTS OF SOURCE PLANTS OF ŌRILA ALONG WITH ITS MARKET SAMPLES. A. TLC PROFILE OF AT UV 254 NM. B. TLC PROFILE OF AT UV 366 NM. C. TLC PROFILE AFTER DERIVATISATION. 1. PLEUROLOBUS GANGETICUS ROOT, 2. PICUROLOBUS GANGETICUS STEM, 3. THIRUVANANTHAPURAM MARKET SAMPLE, 4. IDUKKI MARKET SAMPLE; 5. PALAKKAD MARKET SAMPLE: 6. MALAPPURAM MARKET SAMPLE; 7. KOZHIKODE MARKET SAMPLE; 8. WAYANAD MARKET SAMPLE, 9. KASARGOD MARKET SAMPLE; 10. URARIA PICTA STEM

The HPTLC profile of P. gangeticus root water extract at UV 254nm showed 8 bands and 4 bands in stem.4 bands in Idukki sample. 2 bands in Trivandrum, Malappuram, Wayanad, one band eachin Palakkad and Kasargod samples, 3 bands in Kozhikode sample, 2 bands each in Trivandrum and Idukki samples. Among these samples, 2 bands in Trivandrum and Idukki, Kozhikode, Wayanad and one band each in remaining all samples were found similar to P. gangeticus root. When Rf values at UV 366nm is compared 2 bands in Malappuram sample and one each in all the other samples were found similar to P. gangeticus root and at 550 nm 3 bands in Trivandrum and Wayanad samples, 2 bands in Idukkisample, 1 band each in kozhikode, Wayanad and Malappuram samples were found similar to P. gangeticus root. 3 bands in Trivandrum sample, 2 each in Idukki and Palakkad samples and 1 band in all other sample were found similar to P. gangeticus stem. The details of the HPTLC profile aregiven in the Table 3.

FIG. 15: HPTLC DENSITOMETRIC COMPARISON OF THE HOT WATER EXTRACTS OF SOURCE PLANTS OFŌRILA ALONG WITH ITS MARKET SAMPLES. A. PROFILE OF AT UV 254 NM. B. PROFILE OF AT UV 366 AM C. PROFILE AFTER DERIVATISATION. 1. PLEUROLOBUS GANGETICUS ROOT; 2. PLEUROLOBUS GANGETICUS STEM; 3. THIRUVANANTHAPURAM MARKET SAMPLE: 4. IDUKKI MARKET SAMPLE, 5. PALAKKAD MARKET SAMPLE: 6. MALAPPURAM MARKET SAMPLE: 7. KOZHIKODE MARKET SAMPLE; 8. WAYANAD MARKET SAMPLE, 9. KASARGOD MARKET SAMPLE; 10. URURIA PICTA STEM

The R_f value of the genuine source plants and the market samples of Ōrila were calculated and colour of the bands were also noted. The R_f values at 254nm, 366nm & R_f value visualized after derivatisation are shown in the tables.

TABLE 3: HPTLC PROFILE- THIN LAYER CHROMATOGRAPHY

*P.g – Pleurolobus gangeticus, T- Thiruvananthapuram, I- Idukki, P- Palakkad, M- Malappuram, KZ-Kozhikode, W- Wayanad, K- Kasaragod, U. picta – Uraria picta, Rt- Root, St- Stem.

CONCLUSION: After subjecting all the samples to pharmacognostic investigation like macroscopic, microscopic, histochemical, powder microscopic and HPTLC characteristics as per the standards and comparing those in all parameters we could not find that even one pure sample of Ōrila in market. Macroscopic features are not sufficient for confirming the identity of samples as we got mixtures of samples from almost all the markets out of which some are finely chopped so that macroscopic features are not clearly distinguishable. As per microscopic studies Samples from Trivandrum, Kozhikode and Wayanad contains P. gangeticus roots and stems along with other similar looking plants. Samples from Idukki, Palakkad and Kasargod were mixtures of some similar looking species of Desmodium. Malappuram sample contains roots and stems of Mūvila i.e., Pseudarthria viscida. As per HPTLC analysis also similar bands most matching with Pleurolobus gangeticusroots and stems were observed in Trivandrum, Kozhikode and Wayanad sample but showed some extra bands and differences as the samples were mixture of other plants also. All other sample extracts showed dissimilar bands in UV 254, 366 and Visible lights. And, the price has been rising 10 times during the last 13 years.

ACKNOWLEDEMENTS: The authors are thankful to Dr. Manoj Kumar, Professor and HOD; Dr. Vivek P, Associate Professor; Dr. Jyolsna G Krishna, & Dr. Vidhya Unnikrishnan Assistant professor; Dept. of Dravyaguna Vijnana, V.P.S.V Ayurveda College, Kottakkal, & Miss. Haritha, Technical Assistant, Pharmacognosy Division; Mr. Deepak and Mr Salman Phytochemistry Division; Centre for Medicinal Plants Research Arya Vaidya Sala, Kottakkal for their suggestions, help and support.

Financial Support and Sponsorship: Nil

CONFLICTS OF INTEREST: There is no conflict of interest

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   How to cite this article:

   Athira AA, Madhu KP and Harinarayanan CM: Quality analysis of market samples of ōrila collected from different markets of Kerala using pharmacognostic tools. Int J Pharm Sci & Res 2023; 14(12): 5944-64. doi: 10.13040/IJPSR.0975-8232.14(12).5944-64.

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