Camellia sinensis (leaf)

Nomenclature

Camellia sinensis (L.) Kuntze   Theaceae  
Syn. Thea sinensis L.  
Standardized common name (English): tea

Botanical Voucher Specimen

Source: MOBOT, Tropicos.org.	Source: MOBOT, Tropicos.org.	Source: MOBOT, Tropicos.org.	Source: Royal Botanic Gardens, Kew.

Organoleptic Characteristics

Aroma/Odor: Characteristic Flavor/Taste: Drying, astringent Source: American Herbal Products Association. March 2013. Organoleptic Analysis of Herbal Ingredients. AHPA: Silver Spring, MD

Macroscopic Characteristics

"Youngest leaves narrow, downy, and but slightly serrated. Leaves next in age and size delicately serrated, but venation little perceptible. Leaves of medium and large sizes strongly, deeply, and widely serrated, with well-marked venation, a series of characteristic loops being formed along each margin of the leaves." Source: Clayton et al. Compendium of food microscopy (1909). Tea plantation, Kerala, India (February 2009) Source: Encyclopedia of Life http://eol.org/data_objects/2445507 Fresh leaf, Honde Valley, Zimbabwe (December 2010) Source: Encyclopedia of Life http://eol.org/data_objects/19242832 Flower, Sun Moon Lake, central Taiwan (March 2009) Source: Encyclopedia of Life http://eol.org/data_objects/25801092 Fruit, Honde Valley, Zimbabwe (December 2010) Source: Encyclopedia of Life http://eol.org/data_objects/19242831 Source: Köhler, Medizinal-Pflanzen in naturgetreuen Abbildungen und kurz erläuterndem Texte (1887) Macroscopic image of cut and sifted Camellia sinensis Source: Elan M. Sudberg, Alkemist Laboratories http://www.alkemist.com

Microscopic Characteristics

"1. Upper epidermal cells small and only slightly angular, in leaf of medium size; but larger, more angular, and with walls more distinctly visible, in the old and hard leaf. Hairs and stomata absent. Parenchymal cells similar to those of most other leaves, and not very distinctive. 2. Cells of the lower epidermis larger than those of the upper surface, and associated with stomata and hairs. Stomata, oval or sometimes nearly round, formed of two reniform cells (guard cells) encircling a very apparent aperture; rather numerous, and confined to the under surface of the leaves. The epidermal cells are themselves curved in the neighborhood of the stomata. Hairs short, pointed, and undivided; confined to the under surface of the leaf: very numerous on young leaves, less abundant on old leaves. Wood fibre not characteristic." Source: Clayton et al. Compendium of food microscopy (1909). Transverse Section: “…The upper epidermis is composed of cells with undulating walls and covered with a rather thick cuticle. The lower epidermis consists of smaller cells and is alone provided with stomata; the latter are surrounded by three or four tangentially elongated cells. Simple hairs occur on both surfaces of the leaf, but they are more abundant on the lower; the number, however, varies with the variety of tea, and with the age of the leaf; they are unicellular, tapering and rather thick walled, varying very much in length, but often attaining 500-700 microns. The mesophyll is heterogeneous and asymmetrical. It is characterized by the presence of a large number of sclerenchymatous idioblasts. These are more or less branched and warty and often extend transversely from the upper to the lower epidermis. They vary much in shape and in the thickness of the walls. The cells of the spongy parenchyma contain cluster crystals of calcium oxalate. The midrib is biconvex. Under each epidermis there is a layer of collenchyma of varying thickness. The wood is arched and the bast contains crystals of calcium oxalate. The meristele is surrounded by a pericycle consisting of slightly lignified cells arranged in circle. The cortical tissue contains idioblasts which are usually rather larger and more branched than those of the mesophyll. The little fragments of the stems, which are often to be found in ordinary tea, have a slightly different structure. The wood in them forms a circle within which there is a pith containing branched idioblasts; these have comparatively thin, pitted walls.” Powder: “…The diagnostic characters of powdered tea are:- The characteristic hairs. The sclerenchymatous idioblasts, especially in petiole and midrib. The stomata surrounded by tangentially elongated cells. The calcium oxalate in cluster crystals.” Source: Greenish, H. et. al. (1908) An Anatomical Atlas of Vegetable Powders Twin calcium oxalate crystals, polarized. 200X glyercin : deionized water solution. Source: Amy Brush, Traditional Medicinals Irregular and branched astrosclereid from Camellia sinensis leaf viewed at 400x with Acidified Chloral Hydrate Soln. Source: Elan M. Sudberg, Alkemist Laboratories Thick walled unicellular trichome viewed at 400x under polarized light with Acidified Chloral Hydrate. Source: Elan M. Sudberg, Alkemist Laboratories Source: Greenish, H. et. al. (1908) An Anatomical Atlas of Vegetable Powders

Liquid Chromatographic Identification

UPLC Method

UPLC Camellia sinensis (leaf) UPLC Extraction Solvent: Acetone & water (80:20) Diluent: 0.5% formic acid in water Test Sample Preparation: Transfer 1 g of ground plant material into a screw cap bottle, add 50 ml of Extraction Solvent, tightly cap, and shake for 4 h in a mechanical shaker at room temperature. Filter about 10 ml of extract using a 0.20 um PTFE membrane filter. Dilute 2.0 ml of filtered solution to 10 mL with Diluent. Column: 100 mm x 2.1 mm, 1.7 um, Waters Acquity BEH C18 Mobile Phase: 0.5% formic acid in acetonitrile (Solution A) and 0.5% formic acid in water (Solution B) Elution: Gradient, see Table below Column Temperature: 30°C Flow rate: See Table below Detection: UV, 274 nm Injection volume: 1.0 uL, maintained at 10°C Needle wash: Acetonitrile Source: Indena S.p.A. Table: Gradient program Time (min) Solution A (%) Solution B (%) Flow Rate (mL/min) 0-0.3 5 95 0.35 0.3-7.0 12.3 87.7 0.35 7.0-12.3 13.8 86.2 0.35 12.3-13.0 95 5 0.4 13.0-14.5 95.5 5 0.4 14.5-14.6 5 95 0.4 14.6-15.5 5 95 0.35

HPLC Method

HPLC Camellia sinensis (leaf) HPLC Extraction Solvent: Acetone, water (80:20) Diluent: 0.05% formic acid in water Test Sample Preparation: Transfer 1 g of ground plant material into a screw cap bottle, add 50 ml of Extraction Solvent, tightly cap, and shake for 4 h in a mechanical shaker at room temperature. Filter about 10 ml of extract using a 0.20 um PTFE membrane filter. Dilute 2.0 ml of filtered solution to 10 mL with Diluent. Column: 15-cm x 4.6-mm, 3 um, YMC-Pack ODS-A Mobile Phase: 0.05% formic acid in water (Solution A), 0.05% formic acid in methanol (Solution B), and acetonitrile (Solution C) Elution: Gradient, see Tables below Column Temperature: 40°C Flow rate: 1.0 mL/min Detection: UV, 274 nm Injection volume: 10 uL Source: Indena S.p.A. Table for HPLC systems with dwell volume ˂ 2.0 mL Time (min) Solution A (%) Solution B (%) Solution C (%) 0.0-5.0 97 0 3 5.0-23.0 67 30 3 23.0-29.0 67 30 3 29.0-30.0 30 67 3 30.0-31.0 30 67 3 31.0-31.5 97 0 3 31.5-36.0 97 0 3 Table for HPLC systems with dwell volume > 4.0 mL Time (min) Solution A (%) Solution B (%) Solution C (%) 0.0-1.0 97 0 3 1.0-19.0 67 30 3 19.0-25.0 67 30 3 25.0-26.0 30 67 3 26.0-27.0 30 67 3 27.0-27.5 97 0 3 27.5-36.0 97 0 3

High Performance Thin Layer Chromatographic Identification

Camellia sinensis HPTLC ID - Vanillin Sulfuric Acid UV 365 nm Green Tea (leaf) (Camellia sinensis) Lane Assignments Lanes, from left to right (Track, Volume, Sample): 2 μL Epicatechin ~0.1% in Methanol 3 μL Camellia sinensis-1 (herb) 3 μL Camellia sinensis-2 (leaf) 3 μL Camellia sinensis-3 (herb) 3 μL Camellia sinensis-3 (herb) 4 μL Camellia sinensis-4 (leaf) 2 μL Camellia sinensis-5 (leaf) 2 μL Epigallocatechin Gallate ~0.1% in Methanol Reference materials used here have been authenticated by macroscopic, microscopic &/or TLC studies according to the reference source cited below held at Alkemists Laboratories, Costa Mesa, CA.  Stationary Phase Silica gel 60, F254, 10 x 10 cm HPTLC plates  Mobile Phase CHCl3: ethyl formate: HCOOH [5/4/1]  Sample Preparation Method 0.3 g + 3ml 70% grain EtOH sonicated + heated @ 50° C ~ 1 hr  Detection Method Vanillin/H2SO4 Reagent -> 110° C 5 min -> UV 365 nm  Reference see Herbal Drugs and Phytopharmaceuticals, Wichtl, M., 1994 Source: Elan M. Sudberg, Alkemist Laboratories

Green Tea (leaf) HPTLC ID - Fast Blue salt B reagent, white RT Green Tea (leaf) (Camellia sinensis) Lane Assignments Lanes, from left to right (Track, Volume, Sample): 5 µL (-)-Epigallocatechin-3-O-gallate 5 µL (-)-Epigallocatechin 5 µL (-)-Epicatechin gallate 5 µL (-)-Epicatechin 1 µL Green Tea leaf 1 1 µL Green Tea leaf 2 1 µL Green Tea leaf 3 1 µL Green Tea leaf 4  Reference Sample(s) Reference:Individually dissolve 1 mg of (-)-epigallocatechin and 1 mg of (-)-epicatechin gallate each in 20 mL of methanol; Optional: Individually dissolve 1 mg of (-)-epigallo-catechin-3-O-gallate and 1 mg of (-)-epicatechin each in 20 mL of methanol; Store all solutions at -20°C.  Stationary Phase Stationary phase, i.e. Silica gel 60, F254  Mobile Phase Toluene, acetone, formic acid 9:9:2 (v/v/v)  Sample Preparation Method Sample: Mix 100 mg of powdered sample with 10 mL of methanol, water 4:1 and sonicate for 10 minutes, then centrifuge or filter the solutions and use the supernatants / filtrates as test solutions. Derivatization reagent: Fast Blue salt B reagent; Preparation: dissolve 140 mg of Fast Blue salt B in 10 mL of water and add 140 mL of methanol and 50 mL of dichloromethane. Store reagent in the dark at 4°C; Use: preheat the plate to 100°C for 2 min, then dip (time 0, speed 5), dry for 5 min in the fume hood.  Detection Method Unsaturated chamber; developing distance 60 mm from lower edge; relative humidity 33%  Other Notes Images presented in this entry are examples and are not intended to be used as basis for setting specifications for quality control purposes. System suitability test: (-)-Epigallocatechin: brown zone at Rf ~ 0.46; (-)-Epicatechin gallate: brown zone at Rf ~ 0.52 Identification: Compare result with reference images. The fingerprint of the test solution is similar to that of the corresponding botanical reference sample. Additional weak zones may be present. The chromatogram of the test solution shows four brownish-orange zones corresponding to reference substance epigallocatechin-3-O-gallate (Rf ~ 0.37), (-)-epigallocatechin (Rf ~ 0.46), (-)-epicatechin gallate (Rf ~ 0.52), and (-)-epicatechin (Rf ~ 0.62). The lowest zone is the most intense and the upper zone is the faintest. The two zones in between are clearly separated (black arrows). Source: HPTLC Association