Pharmacognostical Evaluation of an Extra-pharmacopoeial Drug of Ayurveda - Anisochilus carnosus (L.f.) Wall.

Jose, Mallya, Tantrady, Vishwanatha, Prabhu, and Koppala Narayana: Pharmacognostical Evaluation of an Extra-pharmacopoeial Drug of Ayurveda - Anisochilus carnosus (L.f.) Wall.

Authors

INTRODUCTION

India is a rich treasure trove of diverse flora along with ethno-medicinal knowledge. Ethno medicine is the study or comparison of the traditional medicine practiced by various ethnic groups.[1] Traditional knowledge encompasses wisdom, knowledge, teaching and experience of these communities and many a times it is orally transmitted from generation to generation.[2] Urbanization and changed life style resulted in slow erosion of such knowledge rays. India with huge repository of such knowledge must collect and scientifically document these information to enrich its existing Pharmacopeia.[3]

Anisochilus carnosus (L.f) Wall. is an aromatic herb, belonging to family Lamiaceae commonly known as Arikal tumbe by traditional practitioners of Udupi district.[4] It is an annual herb which grows at high altitudes of Western Ghats, fairly common in the crevices of exposed rocks.[5] It is 30 to 60 cm tall, rough, branched plant with leaf stalks upto 1.3 to 5 cm long. Leaves are ovate-oblong, velvety in nature having crenulate margins, inflorescence is a verticillaster. Leaves and aerials parts are used in medicine by traditional practitioners in the treatment of gastric ulcers and stomachache. The leaves are also kept between toes to cure fungal infections of foot and toes.[6] Till now the quality standards of this multiple beneficiary plant has not been documented. With this background, a detailed pharmacognostic study of different parts of the drug employing macro-microscopy, physicochemical standardisation, secondary metabolite screening and HPTLC fingerprinting has been planned in this communication.

MATERIALS AND METHODS

Collection and authentication

Mature whole plant of A. carnosus (L.f.) Wall. flowering and fruiting was collected from Udupi District. Morphology features were compared with Flora of Udupi and a reference sample is deposited at Pharmacognosy Unit of SDM Centre for research in Ayurveda and Allied Sciences Udupi (Vouncher No: 16081201). Plant parts were cleaned from extraneous matter, washed properly in slow tap water before shade drying of root, stem and leaves. After complete air drying the plant material was powdered and preserved for further study. Some fresh samples were preserved in FAA solution for microscopic study.[7]

Macroscopic evaluation

Morphological characters of root, stem and leaves were studied by visual observation following the Standard protocols. These samples were keenly observed under naked eye to record the specific botanical characters and it was also recorded using Canon digital camera with size indicating rulers.[8]

Microscopic evaluation

The histology of root, stem and leaves was recorded following standard procedures. The preserved specimens were cut into thin transverse section using a sharp blade and the sections were stained with saffranine. Transverse sections were photographed using Zeiss AXIO trinocular microscope attached with Zeiss AxioCam camera under bright field light. Magnification of the figures was indicated by the scale-bars.[9]

Powder microscopy

A pinch of powder was warmed with drops of chloral hyadrate on a microscopic slide and mounted in glycerin. Slides were observed under the microscope, diagnostic characters marked and photographed with Zeiss Axio Cam camera under bright field light.[10]

Physico-chemical standards

A.carnosus whole plant powder was tested for pharmacopoeial constants like loss on drying at 105°C, total ash, acid insoluble ash, alcohol soluble extractive, water soluble extractive as per standard protocol.[11]

Preliminary phytochemical analysis

Preliminary phytochemical investigation was done to detect the presence of alkaloids, steroids, carbohydrates, tannin, flavinoids, saponins, triterpenoids, coumarins and phenols in ethanololic extracts of A. carnosus.[12]

HPTLC finger printing

One gram of A. carnosus Whole plant powder was extracted with 10 ml of ethanol. Four, 8 and 12 μl of the above extract were applied on a pre-coated silica gel F254 on aluminum plates to a band width of 7 mm using Linomat 5 TLC applicator. The plate was developed in toluene: ethyl acetate (7.0: 1.0) mobile phasse. The developed plates were visualized under short and long UV and scanned under UV 254nm, 366nm and 620nm before derivatisation with vanillin sulphuric acid. Rf, colour of the spots and densitometric scan were recorded.[13,14]

RESULTS AND DISCUSSION

A. carnosus is an aromatic semisucculent annual herb. Thick tap root, with brownisy white cork layer and aromatic odour. Stems are robust and branched. Leaves 2 to 5 × 2 to 3.5 cm broadly ovate-oblong to circular, base cordate, tip blunt to rounded, petiolate, with crenulate border and a velvety appearance (Fig. 1).

Figure 1

Macroscopy of Anisochilus carnosus (L.f) Wall.

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TS of root

In transverse section root showed outer cork which has thick wall and brown in colour followed by 5 to 6 layerd cortex. Rosette crystals are seen in the cortical region. Vascular bundle is collaterally closed. Large xylem vessels, surrounded by xylem rays are seen. Compared to cortical region, pith region showed big parenchyma cells (Fig. 2).

TS of stem

TS of stem showed trichomes attached to epidermis, beneath which 2 to 3 layers of compactly arranged collenchyma cells are seen. There is a layer of pericycle below the cortex which is mainly composed of parenchyma cells containing starch grain. Endodermis single layered below which there was a conjoint collaterall closed vascular bundle with xylem on the inner side and phloem on the outer side. Pith consisted of bigger parenchyma cells with thick walls (Fig. 3).

TS of leaf

Transverse section of the leaf showed upper epidermis of single layer with cuticle walls and covering type of uniseriate trichomes with blunt apex. Mesophyll was differentiated into palisade and spongy parenchyma. Single layered palisade parenchyma made up of radially elongated cells present. Spongy parenchyma had many layered loosely arranged cells.

Lower epidermis shows numerous trichomes which are unicellular, multicelluar and glandular. In midrib, there was a strip of collenchyma, present below the upper and lower epidermii. This is followed by cortical parenchyma containing crystals of calcium oxalate embedded in the form of rosette crystals. Bicollateral vascular bundle with xylem in the centre and phloem on both the sides are present (Fig. 4).

Figure 2

Microscopy of Root of Anisochilus carnosus (L.f.) Wall.

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Figure 3

Microscopy of Stem of Anisochilus carnosus (L.f.) Wall.

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Figure 4

Microscopy of Leaf of Anisochilus carnosus (L.f.) Wall.

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Powder

Powder showed the epidermal cells with stomata, covering type of both uniseriate and multiseriate trichomes with blunt apex, covering type of uniseriate elongated palisade cells with chlorophyll, bundle of phloem fibres and groups of starch grains (Fig. 5).

Physico-chemical analysis

A. carnosus whole plant powder was tested for loss on drying at 1050C, total ash, acid insoluble ash, ethanol and water soluble extractive as per standard protocol. Loss on drying was 15.43%w/w, total ash was 9.70%w/w, acid insoluble ash 0.30%w/w, water soluble ash 3.96%w/w, alcohol soluble extractive value 5.24%w/w and water soluble extractive value 22.18%w/w. Physicochemical standards were a representative of its purity, physical nature and chemical trait (Table 1).

Phytochemical study

The preliminary photochemical studies are essential to know the basic constituents present in the drug. Action of any drug depends upon these basic components. Preliminary photochemical test were conducted for A. carnosus. Test for alkaloids (Dragendrof’s test, Wagner’s test, Mayer’s test and Hager’s test), carbohydrates (Molisch’s test, Fehling’s test and Benedict’s), steroids (Libermann-Burchard and Salkowski), saponins, phenols, coumarin, triterpenoids, quinine, resin and tannins were conducted and result displayed(Table 2).

HPTLC

HPTLC finger print profile of ethanolic extract of A.carnosus has been obtained with suitable solvent system. The developed plates were visualized under UV light and white and then under light after derivation with vanillin sulphuric acid reagent. Rf, colour of the spots and densitometric scan at 254 and 366 nm were recorded. On photo documentation there were 6 spot under short UV, 13 spots under long UV and 9 spots after derivatisation with vanillin sulphuric acid reagent (Table 3 and Fig. 6). Densitometric scan at 254 nm showed 7 peaks such as 0.01 Rf,(40.78%) 0.13 Rf, (14.97%), 0.27 Rf, (4.89%), 0.40 Rf, (27.25%), 0.58 Rf, (6.70%), 0.64 Rf, (3.76%), 0.76 Rf, (1.64%) (Fig. 6). Densitometric scan at 366 nm showed 11 peak (Fig. 6) whereas at 620 nm there were 8 peaks (Fig. 6).

Figure 5

Powder microscopy of whole plant of Anisochilus carnosus (L.f.) Wall.

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Table 1

Physicochemical standard of whole plant Anisochilus carnosus (L.f). Wall.

ParameterResults n = 3 %w/w
Loss on drying15.43
Total Ash9.70
Acid Insoluble Ash0.30
Water soluble Ash3.96
Alcohol soluble extractive value5.24
Water soluble extractive value22.18
Table 2

Preliminary phytochemical tests of aqueous extract of whole plant Anisochilus carnosus (L.f). Wall.

TestsColour if positiveWater extract
Alkaloids  
Dragendroff’s testOrange red precipitateOrange red precipitate
Wagners testReddish brown precipitateReddish brown precipitate
Mayers testDull white precipitateDull white precipitate
Hagers testYellow precipitateYellow precipitate
Steroids  
Liebermann-buchard testBluish green colourBluish green colour
Salkowski testBluish red to cherry red color in chloroform layer and green fluorescence in acid layerBluish red to cherry red color in chloroform layer and green fluorescence in acid layer
Carbohydrate  
Molish testViolet ringViolet ring
Fehlings testBrick red precipitateBrick red precipitate
Benedicts testRed precipitateRed precipitate
Tannin  
With FeCl3Dark blue or green or brownBrown color
Flavanoids  
Shinoda’s testRed or pinkGreen color
Saponins  
With NaHCO3Stable frothNo froth
Triterpenoids  
Tin and thionyl chloride testPink/Red colorGreen color
Coumarins  
With 2 N NaOHYellowBrown color
Phenols  
With alcoholic ferric chlorideBlue to blue black or brown colorBrown color
Carboxylic acid  
With water and NaHCO3Brisk effervescenceBrisk effervescence
Amino acid  
With ninhydrine reagentPurple colourGreen color
Resin  
With aqueous acetoneTurbidityNo turbidity
Quinone  
Conc. sulphuric acidPink/purple/redRed color
Table 3

Rf values of whole plant Anisochilus carnosus (L.f). Wall.

Under short UVUnder long UVUnder white light after derivatisation
-0.08 (F. red)0.08 (L. purple)
0.11 (L. green)--
-0.13 (F. red)-
--0.16 (L. purple)
-0.19 (F. red)-
--0.21 (D. purple)
-0.29 (F. red)-
--0.31 (L. purple)
-0.33 (F. red)-
0.39 (L. green)0.39 (F. red)0.39 (D. purple)
0.43 (D. green)--
-0.45 (F. red)-
--0.49 (L. purple)
0.52 (L. green)0.52 (F. red)-
-0.54 (F. red)-
0.57 (L. green)-0.57 (D. purple)
-0.63 (F. red)-
0.70 (L. green)0.70 (F. red)-
--0.73 (L. purple)
--0.79 (L. purple)
-0.86 (F. blue)-
-0.92 (F. red)-

*F – fluorescent; L –light; D – dark

Solvent system – Toluene: Ethyl Acetate (7.0: 1.0)

The macroscopic features recorded can be used for preliminary identification of the particular plant. In many of the earlier studies, the microscopic observation of cellular structures was found to be essential. Micorscopic recordings proved to be effective in establishing the authenticity and detection of adulteration/ substitutes for herbal raw drugs. A. carnosus is annual, erect herb. Microscopic features revealed the prescence of rosette crystals in the cortex region of root. Epidermal trichomes and conjoint vascular bundles are anatomical findings of stem. Bicollateral vascular bundles, strips of collenchymatous cells at midrib and epidermal unicellular, multicellular, gladlular trichomes are main features of leaf anatomy. Uniseriate and multiseriate trichomes along with groups of starch grains are marked features of powder microscopy.

Any matter other than the described parts of the drug is to be considered as foreign matter, any raw drug must be made free from foreign matter before any physico-chemical analysis is done.Total ash indicative of the total inorganic composition of the drug was found to be 9.70% w/w, acid insoluble ash indicating the silicacious matters was found to be 0.30% w/w. Water soluble ash which indicates the amount of ash which is readily soluble in water was found to be 3.96% w/w. Loss on drying indicates the moisture and volatile matter content in sample and was found to be 15.43% w/w. The solvent used for the extraction is in a position to dissolve appreciable quantities of substances likewise various solvents were used to extract these chemical constituents. The extract obtained by percolating coarse powder is indicative of approximate quantity of their chemical constituents. Alcohol soluble extractive value of the test sample was found to be 5.24% w/w and water soluble was 22.18% w/w. All these pharmacopoeial parameter helps to determine the quality and purity of herbal drugs. Preliminary phytochemical tests were conducted using the water extracts alkaloids, Steroids, Carbohydrate, Tannin, Phenols, Carboxylic acid, and Quinone. These preliminary analyses of chemical composition are one of the primary methods to study the chemistry of herbs. HPTLC photo documentation revealed presence of phyto-constituents with different Rf values. Densitometric scan of the plates showed diagnostic bands under 254 nm, 366 nm and post derivatisation. HPTLC finger printing is an effective technique of screening herbal raw drugs for authenticity and quality.

Figure 6

HPTLC of ethanolic extract of whole plant of Anisochilus carnosus (L.f.) Wall.

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CONCLUSION

In Ayurvedic classics, it is advised to learn about the plants from natives and tribes, from shepherds, hermits and other experts who have well versed knowledge about these plants. Traditional knowledge is the hidden source of knowledge. A. carnosus (L.f) Wall. a member of Lamiaceae family is a less known drug, but used by the traditional Vaidyas for Finger-gap infections and other skin diseases. A. carnosus an annual aromatic herb with quadrangular stem commonly called as Arikal tumbe, the aerial parts of which are used in many therapeutic conditions. In transverse section root showed outer cork which was thick wall brown in colour followed by cortex 5-6 layers. Anatomical features of Stem of A. carnosus exhibited trichomes, which were attached to epidermis; beneath this 2 or 3 layers compactly arranged collenchymas was present. Microscopic characters of leaf shows upper epidermis single layer with cuticle cyst walls, covering of trichomes which were uniserate with blunt apex. Powder microscopy characteristics showed the presence of starch in parenchyma region. Physicochemical standards and HPTLC represent the standard outprints of the drug whereas preliminary phytochemical study of alcoholic extract showed it to possess alkaloids, carbohydrates, steroids, tannins, phenol, carboxylic acid and quinone.

GRAPHICAL ABSTRACT

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ACKNOWLEDGEMENT

Authors are grateful to revered President, Dr. D. Veerendra Heggade, SDM Educational Society for constant his encouragement. Authors are indebted to Dr. Shreekanth U., Principal SDM Udupi and Dr. B. Ravishankar, Research Director SDM Centre for Research in Ayurveda and Allied Sciences, Udupi for their support.

ABOUT AUTHOR/S

Dr. Dintu Jose BAMS, MD scholar at Department of Dravyagauna, SDM College of Ayurveda Kuthpady ,Udupi. Her field of interest, are antimicrobial study of herbal drugs, documentation of traditional knowledge. The whole research work was carried out by this scholar, collection of plant material, pharmacognostic, phytochemical study was carried out by the scholar, report collected and arranged systematically.

Dr. Suma V. Mallya MD, PhD (Ayu, Dravyagauna) a practicing Ayurvedic physician and Associate professor at SDM College of Ayurveda , Kuthpady, Udupi. Obtained her PhD from MUHS Nashik. Recognized PG and PhD guide in Dravyagauna from RGUHS Bangalore. She has presented many papers in national and international seminars and published around 15 scientific papers in various journals. SVM is research guide for this work, actual planning, advices, timely suggestions were given by her and the paper was prepared, and corresponded for publication.

Dr. Bairi Shridhara MD, PhD (Ayu, Dravyaguna) practicing Ayurvedic physician and former HOD and professor, Department of Dravyaguna, SDM College of Ayurveda, Kuthpady, Udupi. He was a former director for Folklore research centre, SDM Udupi, and has published many research articles. Has guided many PG and Phd students of Ayurveda. The plant information, traditional medicinal uses and collection of plant material was carried out under his guidance.

Dr. Vishwanatha, MSc, PhD, Research officer at Microbiology and biotechnology department, at SDM Ayurveda and Allied Sciences, Udupi, India 574118. Published many scientific papers at national and international Journals. He is presently working on VGST and other projects. VU is co-guide for this work.

Suchitra Prabhu, Research officer at Pharmacognosy and phytochemistry department, at SDM Ayurveda and Allied Sciences, Udupi, India 574118.She has published many scientific papers at national and international Journals. SP assist in providing pharmacognostic and phytochemical study reports.

Dr. KN Sunil Kumar PhD is working as Research Officer and Head of Department of Pharmacognosy in Siddha Central Research Institute, Chennai, India. He has completed in M.Sc. Ayu. Medicinal Plants -Pharmacognosy specialization from Gujarat Ayurveda University in 2006 and PhD in Medicinal Plants/Pharmacognosy from University of Madras in 2014. His field of expertise includes Taxonomy, Pharmacognosy, Phytochemistry, Standardization, Quality control, Pharmacology and In vitro assays. He has published 92 research papers in national and international peer reviewed journals in addition to 55 monographs in Quality standards of Indian Medicinal Plants. Guidance and supervision the experiments until finalization of the paper was undertaken by KNSK.

Notes

[2] Conflicts of interest CONFLICT OF INTEREST Nil

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