INTRODUCTION:

Coptis teeta Wall. belongs to the family Ranunculaceae and is also known as golden thread herb, Mamira and Mishmi. It is an endangered species in India. C. teeta a small perennial herb is an important medicinal plant used for various treatments of various diseases. C. teeta has been reported only from Indian Territory of Arunachal Pradesh and Yunnan Province of China.^1-2 The plant has various indications in the traditional System of Medicine i.e., Ayurveda, Unani, Siddha and Chinese system.

The demand for the root of C. teeta is very high, especially in Asian countries. The root of this plant is medicinally useful and has to be uprooted for the collection. It is responsible for the critical condition of this plant. This herb is distributed from 1700 m to 2800 m, especially in the Himalayan region.³ It is a perennial stemless herb with horizontal rootstocks and densely fibrous with yellowish brown externally and golden-yellow internally and very bitter. Leaves are pinnatifid with long petioles; ovate-lanceolate leaflets are shiny and slippery. Flowers are white or yellowish with stalked follicles.⁴C. teeta is widely used by Mishmi and other tribes of Arunachal Pradesh for a variety of health issues like malaria, dysentery, cold and cough, diarrhea, blood dysentery, typhoid, hypertension, hepatitis, febrifuge, stomach pain and also to relieve pain of eyes conjunctivitis.^2-,5 Roots contains berberine and it acts as a bitter tonic. Due to several alkaloids rhizomes is bitter (Berberine, Coptisine and Palmatine, etc.) which are useful in preventing various microbes and also in treatment for many disorders.⁶ Berberine has the property to treat type 2 diabetes, reduce and maintain blood sugar levels, and lower cholesterol levels through a mechanism different than statin drugs.^7,8 There is a great chance of adulteration of Coptis teeta Wall due to its limited and rare availability in India. It has been thought worthwhile to undertake the Pharmacognostic and phytochemical evaluation of Coptis teeta Wall.) for its identity, purity, and strength.

MATERIALS AND METHODS:

Chemicals: Only analytical grade chemicals were used in studies. The list of chemicals is as follows: Phloroglucinol (LOBA Chemie), Safranin (LOBA Chemie), Fast green (LOBA Chemie), Dragendroff’s solution (LOBA Chemie), Hydrochloric acid (s.d.fine CHEM LTD), Sulphuric acid (s.d.fine CHEM LTD), Sodium hydroxide (s.d.fine CHEM LTD), Sudan red III (LOBA Chemie), Ruthenium red (s.d.fine CHEM LTD), Ferric chloride (s.d.fine CHEM LTD), Alcohol (CS), Acetic acid (s.d.fine CHEM LTD) and Iodine solution (Thomas Bakers). Instruments: Stereo microscope LEICA S9D and EC4 camera, Olympus microscope BX 43 and LC camera 30 and Nikon D7200 camera, CAMAG linomat V applicator and CAMAG UV visualization assemble.

Plant materials: The roots of C. teeta were collected from the Naharlagun, Arunachal dated 20/01/2019. The plant was authenticated by Dr. Shiddamallayya N, Research Officer (Botany)⁹, A voucher specimen of the plant was preserved in the herbarium section of R.A.R.I. Itanagar. The collected drug is kept in the Pharmacognosy section of RARI, Pune (Ref. No. IMRPCG2018-2019-17).

Macroscopic and Organoleptic Analysis:

Macroscopic characters and organoleptic characters of C. teeta root were observed and noted.¹⁰

Microscopic characterization:

Freehand sections (T.S.) of a root were taken. The section was stained using 0.1% w/v solution of safranin dye. The Image of T.S. was taken using Olympus BX 43 and LC 30 camera.¹¹

Powder microscopy:

The sample of powder (1mg) was stained with Iodine solution (composed of 2g iodine and 3g potassium iodide in 100mL of water) and then mounted in 50% glycerine on one slide. On another slide, the sample of 1 mg was stained with phloroglucinol (0.1% w/v) and dilute Hcl (10% solution) then washed in water and after that mounted in 50% glycerine. The microscopical characteristics of both the prepared slides were observed using Olympus BX 43 and LC 30 camera.¹²

Histochemistry:

Histochemical evaluation of leaves was conducted as per the standard procedure.¹³

Fluorescence analysis:

Powdered leaf material was analyzed under visible light and ultraviolet light after treatment with various organic/inorganic reagents.¹⁴

Physiochemical analysis:

Physicochemical analyses such as loss on drying (LOD), alcohol soluble extractive values, water-soluble extractive values, total ash value, acid-insoluble ash, and pH were carried out as per the method described by Ayurvedic Pharmacopoeia of India.¹⁵

Preliminary phytochemical screening:

Alcoholic and aqueous extracts of C. teeta root were prepared by cold maceration method. Coarsely powdered air-dried material 5g was accurately weighed and placed in a glass stoppered conical flask. It was macerated with 100ml of the solvent specified for 6hr, shaken frequently and then allowed to stand for 18hr. Filtered rapidly taking care not to lose any solvent, the filtrate was transferred to a tared flat-bottomed dish and evaporated to dryness on a digital water bath at 60° C. Both the extracts were subjected to phytochemical screening for qualitative analysis for the presence and absence of secondary metabolites.¹⁶

HPTLC:

1g drug powder was taken in 10ml methanol in a test tube, heated in a water bath for 15 min at 60°C, and then filtered. The filtrate (4µl) was applied as spot on TLC silica gel 60 F254 aluminum coated plates using the Linomat V applicator of CAMAG. The process was repeated 4 times for validation of the result. The TLC plate was developed in CAMAG TLC chamber 10x20 using mobile phase (Ethyl acetate: Butanol: acetic acid: water (5:3:1:1). The developed plate was seen in CAMAG UV chamber at 254 and 366nm. The plate was also visualized in diffused sunlight.¹⁷

RESULTS AND DISCUSSIONS:

Detail macroscopic and organoleptic characters (Figure 1) are as follows: Shape - Un-straight, Irregular, Size – 3 to 6cm in length and 1 to 2cm in width, Fracture – Irregular, Colour - Outer surface- Brownish, Inner surface–yellowish, Odour-Characteristic, Taste-Bitter, Surface–Rough. Texture-outer surface-rough and wrinkled, inner surface-perforated.

Transverse section of root showing (Figure 2) thick layered periderm, well-differentiated into phellem, phellogen and phelloderm measuring about 162µm in length; phelleme cells are sub-rectangular and thick-walled; phellogen cells are brick-shaped and single layered; phelloderm cells are 2-3 layered, sub- rounded. The cortex is divided into outer and inner; outer cortex cells are oval, aerenchymatous which is deposited with berberine and starch grains; inner cortex cells are oval, collenchymatous, thin-walled, which is deposited with berberine, starch grains. Secondary phloem cells are not organized, somewhat oval cells, non-lignified cells. The vascular bundle is made up of fibers and xylem present in patches. Pith is made up of parenchymatous, rounded cells deposited with starch grains.

Figure 1: Macroscopic character of the root of C. teeta Wall showing: a. root; b. powder of root; c. to g- zoom image of root using a stereo microscope: - c. protrusion on outer surface; d. wrinkled outer surface; e. internal structure of root in dried sample; f. irregular fracture; g. the internal structure of root in a wet sample

Powder microscopy of root (Figure 3) of C teeta showing fragments of surface view of cork cells. Cork cells show rectangular, thick-walled, highly deposited phellem and polygonal, thick-walled, comparatively smaller than phellem cells, highly deposited phellogen. Outer cortical cells are beaded, rounded, and deposited with berberine and starch grains; they possess air space. Inner cortical cells are rounded, and deposited with berberine and starch grains; it is without air space. Surface view of pith cells showing somewhat oval to sub-rounded. Berberine is embedded inside cortical cells. A sectional view of pith cells showing polygonal, thin-walled, parenchymatous cells. Vascular region showing non-lignified, elongated phloem fibers; xylem with spiral and reticulate thickening. Prism crystals of calcium oxalate with 4-5 angles. Simple and compound rounded starch grains are seen.

Figure 2: T. S. of the root of C. teeta . At 10× 10 Showing (Bark (ba) consists of periderm, secondary cortex, phloem or bast cells; Periderm (pe) consists of phellem (ph), phellogen (pg), phelloderm (pd); Secondary cortex shows outer (osc) and inner cortex (isc), Secondary phloem cells (sph); Xylem cells consists of xylem vessels (xv) and tracheids (xt). pith (pi) at mid of section)

Figure 3: Powder microscopy of the root of C. teeta. Showing (a. Phellem cells in surface view; b. Phellogen cells in surface view; c. Outer cortical; cells; d. Inner cortical cells; e. Surface view of pith cells, f. Berberine inside cortical cells; g. sectional view pith cells; h. Reticulate thickening of xylem; i. Phloem non- lignified fiber; j. Prism crystals of calcium oxalate; k. Spiral thickening of xylem; l. Uncoiled spiral thickening of xylem; m. Simple and compound starch grains.

Histochemical Test:

Powder drug was analyzed in 3 separate batches for histochemical tests The analysis result is depicted in Table -1.

Table 1: Histo-chemical analysis

Sr. No.	Test	Chemical	Observation	Result
1	Alkaloid	Dragendorff's reagent	Brick red	+
2	Lignified cell	Phloroglucinol + HCl	Pink to cherry red colour	+
3	Cuticular cell	Sudan Red -III	-	-
4	Aleurone grains	Iodine	-	-
5	Fats and volatile oils	Sudan Red- III	-	-
6	Mucilage fatty oils	Ruthenium red	-	-
7	Starch	Iodine	Blue or reddish blue	+
8	Calcium oxalate crystals	Hydrochloric acid	Dissolved	+
9	Calcium carbonate crystals	Hydrochloric acid	-	-

+ Present; - Absent

Fluorescence analysis:

The powder drug was analyzed in 3 separate batches for

Fluorescence analysis. The analysis result is depicted in Table -2.

Table 2: Fluorescence analysis

Test	Daylight	254 nm	366 nm
Powder as such	Light sienna	Seinna	Fulvous
Powder + H₂0	Light pale luteous	Pale greenish grey	Slightly pale purplish grey
Powder + HCl	Amber	Olivaceous	Dark herbage green
Powder + HNO₃	Scarlet	Dull green	Dark fulvous
Powder + H₂SO₄	Bay	Umber	Sepia
Powder + GAA	Sulphur yellow	Citrine green	Dark herbage green
Powder +18N HCl	Amber	Greenish glaucous	Greish yellow-green
Powder + 50% HNO₃	Scarlet	Amber	Amber
Powder + 50% H₂SO₄	Amber	Citrine	Yellowish green
Powder + 50% GAA	Amber	Herbage green	Purplish grey
Powder + 1N NaOH	Amber	Amber	Luteous
Powder + 1N KOH	Amber	Amber	Luteous
Powder + 0.1N Iodine	Blood color	Blood color	Fuscous black
Powder + 5% FeCl₃	Dark green	Citrine	Dull green
Powder + Liquid NH₃	Luteous	Herbage green	Umber

Preliminary Phytochemical screening:

Phytochemical screening reveals the presence of phytoconstituents as depicted in Table 3

Table 3: Preliminary Phytochemical Screening

S. No.	Phytoconstituents	Aqueous extract	Alcoholic extract
1	Carbohydrate	+	+
2	Protein	+	+
3	Lipid	-	-
4	Alkaloids	-	+
5	Glycoside	+	+
6	Phenolics	+	+
7	Flavonoids	+	+
8	Tannins	+	+
9	Saponin	-	-
10	Steroids		+
10	Volatile oils	-	-
11	Triterpenoids	-	-

+ Present, - absent

Determination of physicochemical parameters:

Results obtained from physicochemical contents such as loss on drying, total ash, acid insoluble ash, water, and alcohol soluble extracts are depicted in Table 4.

Table 4 - Physico-chemical parameter

S. No	Parameter	Result
1	Loss on drying	Should not be more than 7.30 %
2	Total Ash	Should not be more than 3.69 %
3	Acid insoluble ash	Should not be more than 0.782 %
4	Water soluble extractive	Should not be less than 32.344%
5	Alcohol soluble extractive	Should not be less than 17.963%
The data depicted in the table is the mean of three samples.

Thin Layer Chromatography:

5 spots were seen in UV light at 254nm however no spots were detected in visible light as well as at 366 nm. The R_f value of spots detected at 254nm is as follows: Spot 1- Fuscous black – 0.07, Spot 2- Pale mouse grey- 0.11, Spot 3- Light Pale mouse grey – 0.17, Spot 4- Light fuscous black- 0.23 and Spot 5- Light fuscous black- 0.34). R_f value is a constant that may play an important role in identification as well as ensuring the quality of genuine samples.

The outcome of the current research would provide important quality data for standardization and quality control of the root of Coptis teeta Wall. Morphological and organoleptic assessment of the root of C.teeta imparts a significant role in the identification of the root of C. teeta and detection of adulteration. The microscopic (transverse section) evaluation allows a more detailed examination of a medicinal plant and it can be used to identify the organized drugs by their known histological character. Powder microscopy imparts a crucial role in a similar way as T.S. of root. Standardization of medicinal plants is a complex process so different physicochemical parameters impart an important role in standardization. The physicochemical parameter can be used as a standard to ensure the quality of crude drugs. The Ash value of the root shows the presence of inorganic content. The lower value of the acid-insoluble ash suggests the greater physiological availability of the crude drug. Extractive value gives information about the availability of soluble phytoconstituents in a particular solvent. The low value of moisture content does not encourage microbial contamination as the general requirement of moisture content in the crude drug is not more than 14 % (W/W). TLC plays a crucial role in plant standardization.

CONCLUSION:

Pharmacognostic characters and a physicochemical parameter can be used as standards for the authentication of plants and detection of adulteration.

ACKNOWLEDGEMENT:

The authors are thankful to The Central Council for Research in Ayurvedic Sciences (CCRAS) and, the Ministry of AYUSH Government of India for financial support to the Project.

CONFLICT OF INTEREST:

Nil

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Received on 07.05.2022 Modified on 17.07.2023

Research J. Pharm. and Tech 2024; 17(5):1933-1937.

DOI: 10.52711/0974-360X.2024.00306