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Abstract ［Objectives］ This study was conducted to investigate the quality standard of Heracleum moellendorffii Hance., so as to provide data support for the 2003 version of Quality Standards for Chinese Medicinal Materials and Ethnic Medicinal Materials in Guizhou Province.
［Methods］H. moellendorffii was subjected to character identification and microscopic identification. Referring to Chinese Pharmacopoeia, TCL thin-layer identification and extract determination were carried out on 10 batches of H. moellendorffii from 6 habitats in Guizhou.
［Results］ The characters and microscopic characteristics of H. moellendorffii were described in detail. The TLC thin-layer identification spots were clear, with strong specificity. The limit of the medicinal material extract was determined to be not less than 22.0%.
［Conclusions］This study can provide data support for the quality evaluation and standard improvement of the medicinal material H. moellendorffii.
Key words Heracleum moellendorffii; Identification of crude drugs; Thin-layer identification; Extract
Received: January 2, 2021 Accepted: March 13, 2021
Supported by Science and Technology Innovation Platform Construction Project of Anshun City (ASKP［2017］03).
Pengfei XIA (1986-), male, P. R. China, chief pharmacist, devoted to research about drug inspection and quality standards.
*Corresponding author. E-mail: yusi73@126.com.
The medicinal material of Heracleum moellendorffii Hance. refers to its dried roots and rhizomes［1］, which has been used in folk［2］ as early as the Song Dynasty due to its effects of dispelling wind, removing dampness, and relieving pain, for treating wind-cold-dampness arthralgia, waist and knee pain, Shaoyin Fufeng headache［3］. It has been reported in literatures that the main components of H. moellendorffii are pimpinollinand isopimpinellin and other furanocoumarins［4］. Most of the coumarin compounds show anti-inflammatory, anticoagulant, anti-cancer and other pharmacological activity［5］, which is basically the same as the effect of H. moellendorffii. H. moellendorffii is mainly produced in Yunnan, Guizhou, Sichuan, and Gansu, but the production is small. In some provinces, such as Hunan, although there is distribution, it is not used for medicinal purposes, and only used as regional varieties. H. moellendorffii is included in the 2003 edition of the Quality Standards for Chinese Medicinal Materials and Ethnic Medicinal Materials in Guizhou Province. Although included in the original standards, only character descriptions are included. This study provides data support for the improvement of the quality standards of H. moellendorffii by of microscopy, thin layer identification and extract determination. Materials and Methods
Instruments
DM500 LEICA camera microscope; METTLER TOLEDO balance; KQ3200DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.).
Materials
The materials used included blades, glass slides, coverslips, tweezers, chloral hydrate, phloroglucinol, concentrated hydrochloric acid, dilute glycerin and distilled water for conventional slide preparation, and 10 batches of H. moellendorffii samples, which were washed and cut into thick slices, and sun-dried for later use. The sources and batch numbers of samples are shown in Table 1. The specimens were identified as H. moellendorffii by Professor Wei from the Department of Medicinal Plant Cultivation and Identification, College of Pharmacy, Guizhou University of Traditional Chinese Medicine.
Methods and Results
Pharmacognosy identification
Plant morphology
H. moellendorffii is a kind of perennial herb of 1-2 m high. The root is conical, thick, divergent, gray-brown. The stem is upright, ridged, and branches on the upper part. The leaves are petiolate, 10-30 cm in length; they have a broadly ovate outline, and are thin and ternate; the lobes are broadly ovate to round, heart-shaped, irregular with 3-5 lobules, 10-20 cm long and 7-18 cm wide; and the edges of the lobes have thick serrations, sharp to long pointed, and the petiolule is 3-8 cm long. The upper leaves of the stem have significantly broad leaf sheaths. Compound umbels are terminal and lateral, and the peduncle is 4-15 cm long; there are few involucral bracts, linear-lanceolate; the umbels have a width of 12-30 and unequal lengths; the bracteoles are 5-10 in number, lanceolate; the flower stalk is slender, 4-20 mm long; the calyxteeth are not conspicuous; the petals are white, dimorphic; and the style base is short conical, and the style diverges. The cremocarp is round and obovate, concave at the top, flat at the back, about 8 mm in diameter, sparsely pilose or nearly smooth, and have back and middle ridges linearly protruding and wide lateral ridges; and each furrow has vitta 1 and commissure vitta 2 therein, and is rod-shaped, half the length of the cremocarp, and the endosperm ventral surface is straight. The plant is shown in Fig. 1, and the fruit is shown in Fig. 2.
Character identification
The product is long conical, slightly curved, 15-40 cm in length, and brown or grayish brown in appearance, and has 2-3 or more branches at the lower part. The root head is enlarged, about 1.5-3.0 cm in diameter, and has dense ring patterns and stem and leaf residues or depressions on the top; there are irregular wrinkles and a number of protruding horizontal lenticels at the lower part of the root head, and visible fibrous root marks can be observed; the root is light weigh, and can be broken easily, showing the fracture surface which is not flat, and the skin is white and has many small brown spots; and the xylem is yellow and white. In addition, the product has aroma, and tastes bitter and slightly pungent. The H. moellendorffii medicinal material is shown in Fig. 3. Microscopic identification
Cross section: The cross section of H. moellendorffii is observed to have following microscopic identification features:
The phellem layer is a number of columns of cells, and the phelloderm is narrow. The cortex has a few oil chambers; and the phloem is broad, accounting for more than 1/2 of the entire root, and there are many oil chambers, radially 40-80 μm, tangentially 26-146 μm, surrounded by 6-10 secretory cells. The cambium forms a ring; and the xylem vessels are numerous, 25-52 μm in diameter, single or clustered. The rays are 1-2 rows of cells in width. The parenchyma cells contain starch granules. Fig. 4, Fig. 5, and Fig.6 show the details.
Powder: The powder is light yellow, and shows numerous starch granules. Single granules are quasi-circular, 5-14 μm in diameter, and some show umbilical points, dotted or herringbone. The layered patterns are not obvious, and there are many compound granules. Most of the vessels are reticulated, and 25-52 μm in diameter. The cork cells are brown-yellow. The vittae are mostly broken, and there are brown-yellow secretions in the secretory cells. Fibers are rare, 16-27 μm in diameter. Fig. 7 shows the details.
Thin layer identification
H. moellendorffii from 6 habitats in Guizhou was selected as the test samples. A certain amount of powder (2 g) was added with 10 ml of methanol, obtaining a mixture, which was ultrasonically treated for 15 min and filtered, giving the filtrate as a test solution. A certain amount of the control medicinal material of H. moellendorffii (2 g) was prepared into a control medicinal material solution by the same method. Then, 10 μl of each of the test solutions and the control medicinal material solution were transferred and dotted on the same silica gel G thin-layer plate and developed with petroleum ether (60-90 ℃)-ethyl acetate (7∶3) as the developing agent. After air-drying, the plate was inspected under UV light (365 nm), as shown in Fig. 8.
Determination of extract
H. moellendorffii root powder (Nwdh3) was taken and determined according to the extract determination method in Chinese Pharmacopoeia (2015 edition, general rule 2201). Using different concentrations of ethanol, it can be determined that when dilute ethanol was used as the solvent, the extract content of the Chinese medicinal material, H. moellendorffii roots was higher. The results are shown in Table 2.
Through the selection of the cold soaking method, hot soaking method and extraction solvent, it can be seen that when the hot soaking method used dilute ethanol as the extraction solvent, the content of H. moellendorffii extract was higher. It was tentatively decided to use dilute ethanol as the solvent, and the hot soaking method was used to determine H. moellendorffii extracts. The extracts of 10 batches of H. moellendorffii were determined (calculated in dry product). The results are shown in Table 3. According to the determination results, the extracts of 10 batches of samples were in the range of 22.22-35.51, with an average value of 26.9. It was tentatively decided that the extract standard should not be less than 22.0%.
Pengfei XIA et al. Study on the Quality Standard of Heracleum moellendorffii Hance.
S: Control medicinal material of H. moellendorffii; 1: Leishan County, Southeastern Guizhou; 2: Bayang Town, Ziyun County, Anshun City; 3: Jiucaiping, Bijie City; 4: purchased (Ziyun County, Anshun City); 5: Yangwu Town, Ziyun County, Anshun City; 6: purchased (Anshun City).
Conclusions and Discussion
The quality standards of H. moellendorffii were included in the 2003 edition of Quality Standards for Chinese Medicinal Materials and Ethnic Medicinal Materials in Guizhou Province, but the original standards are relatively simple, and have no detailed descriptions of traits and microscopic identification, and no extract and thin-layer identification experiments, so there are certain limitations in evaluating the quality of medicinal materials. Based on the original standards, in this study, we improved the character and microscopic identification, and added extract and thin-layer identification. Since furanocoumarins reported in the literatures are the main medicinal ingredients, and the National Institute for the Control of Pharmaceutical and Biological Products did not have relevant reference materials, no content determination method were established at this time. In TLC thin-layer identification, the control medicinal material and the samples were developed with different developing solvents including petroleum ether (60-90 ℃)-acetone (7∶3), petroleum ether (60-90 ℃)-ethyl acetate (7∶3) and ethyl acetate-methanol-water (5∶1∶0.5), and examined under 365 nm ultraviolet light, and it was found that with petroleum ether (60-90 ℃)-ethyl acetate (7∶3), the spots were clear in color, and the resolution and repeatability were good. This study can provide a scientific basis for the improvement of the 2003 version of Quality Standards for Chinese Medicinal Materials and Ethnic Medicinal Materials in Guizhou Province.
References
［1］ Editorial Board of Flora of China, Chinese Academy of Sciences. Flora of China［M］. Beijing: Science Press, 1974. (in Chinese)
［2］ SONG PS, ZHU JR, et al. Materia medica study of Chinese herbal medicines Heracleum produced in Gansu ［J］. Acta Chinese Medicine and Pharmacology, 2004, 32(4): 24, 25. (in Chinese)
［3］ Editorial Board of Quality standards for Chinese medicinal materials and ethnic medicinal materials in Guizhou Province. Quality standards for Chinese medicinal materials and ethnic medicinal materials in Guizhou Province［M］. Guiyang: Guizhou Science and Technology Publishing House, 2003. (in Chinese)
［4］ RAO GX, YANG Q, CAI F, et al. Chemical composition of Heracleum moellendorffii Hance.［J］. Journal of Yunnan University of Traditional Chinese Medicine, 1997, 17(3): 4-6. (in Chinese)
［5］ ZHOU LL, ZENG JG. Chemical constituent and pharmacological research progress of Heracleum hemsleyanum Diels［J］. Modern Chinese Medicine, 2019, 21(12): 1739-1748. (in Chinese)
［Methods］H. moellendorffii was subjected to character identification and microscopic identification. Referring to Chinese Pharmacopoeia, TCL thin-layer identification and extract determination were carried out on 10 batches of H. moellendorffii from 6 habitats in Guizhou.
［Results］ The characters and microscopic characteristics of H. moellendorffii were described in detail. The TLC thin-layer identification spots were clear, with strong specificity. The limit of the medicinal material extract was determined to be not less than 22.0%.
［Conclusions］This study can provide data support for the quality evaluation and standard improvement of the medicinal material H. moellendorffii.
Key words Heracleum moellendorffii; Identification of crude drugs; Thin-layer identification; Extract
Received: January 2, 2021 Accepted: March 13, 2021
Supported by Science and Technology Innovation Platform Construction Project of Anshun City (ASKP［2017］03).
Pengfei XIA (1986-), male, P. R. China, chief pharmacist, devoted to research about drug inspection and quality standards.
*Corresponding author. E-mail: yusi73@126.com.
The medicinal material of Heracleum moellendorffii Hance. refers to its dried roots and rhizomes［1］, which has been used in folk［2］ as early as the Song Dynasty due to its effects of dispelling wind, removing dampness, and relieving pain, for treating wind-cold-dampness arthralgia, waist and knee pain, Shaoyin Fufeng headache［3］. It has been reported in literatures that the main components of H. moellendorffii are pimpinollinand isopimpinellin and other furanocoumarins［4］. Most of the coumarin compounds show anti-inflammatory, anticoagulant, anti-cancer and other pharmacological activity［5］, which is basically the same as the effect of H. moellendorffii. H. moellendorffii is mainly produced in Yunnan, Guizhou, Sichuan, and Gansu, but the production is small. In some provinces, such as Hunan, although there is distribution, it is not used for medicinal purposes, and only used as regional varieties. H. moellendorffii is included in the 2003 edition of the Quality Standards for Chinese Medicinal Materials and Ethnic Medicinal Materials in Guizhou Province. Although included in the original standards, only character descriptions are included. This study provides data support for the improvement of the quality standards of H. moellendorffii by of microscopy, thin layer identification and extract determination. Materials and Methods
Instruments
DM500 LEICA camera microscope; METTLER TOLEDO balance; KQ3200DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.).
Materials
The materials used included blades, glass slides, coverslips, tweezers, chloral hydrate, phloroglucinol, concentrated hydrochloric acid, dilute glycerin and distilled water for conventional slide preparation, and 10 batches of H. moellendorffii samples, which were washed and cut into thick slices, and sun-dried for later use. The sources and batch numbers of samples are shown in Table 1. The specimens were identified as H. moellendorffii by Professor Wei from the Department of Medicinal Plant Cultivation and Identification, College of Pharmacy, Guizhou University of Traditional Chinese Medicine.
Methods and Results
Pharmacognosy identification
Plant morphology
H. moellendorffii is a kind of perennial herb of 1-2 m high. The root is conical, thick, divergent, gray-brown. The stem is upright, ridged, and branches on the upper part. The leaves are petiolate, 10-30 cm in length; they have a broadly ovate outline, and are thin and ternate; the lobes are broadly ovate to round, heart-shaped, irregular with 3-5 lobules, 10-20 cm long and 7-18 cm wide; and the edges of the lobes have thick serrations, sharp to long pointed, and the petiolule is 3-8 cm long. The upper leaves of the stem have significantly broad leaf sheaths. Compound umbels are terminal and lateral, and the peduncle is 4-15 cm long; there are few involucral bracts, linear-lanceolate; the umbels have a width of 12-30 and unequal lengths; the bracteoles are 5-10 in number, lanceolate; the flower stalk is slender, 4-20 mm long; the calyxteeth are not conspicuous; the petals are white, dimorphic; and the style base is short conical, and the style diverges. The cremocarp is round and obovate, concave at the top, flat at the back, about 8 mm in diameter, sparsely pilose or nearly smooth, and have back and middle ridges linearly protruding and wide lateral ridges; and each furrow has vitta 1 and commissure vitta 2 therein, and is rod-shaped, half the length of the cremocarp, and the endosperm ventral surface is straight. The plant is shown in Fig. 1, and the fruit is shown in Fig. 2.
Character identification
The product is long conical, slightly curved, 15-40 cm in length, and brown or grayish brown in appearance, and has 2-3 or more branches at the lower part. The root head is enlarged, about 1.5-3.0 cm in diameter, and has dense ring patterns and stem and leaf residues or depressions on the top; there are irregular wrinkles and a number of protruding horizontal lenticels at the lower part of the root head, and visible fibrous root marks can be observed; the root is light weigh, and can be broken easily, showing the fracture surface which is not flat, and the skin is white and has many small brown spots; and the xylem is yellow and white. In addition, the product has aroma, and tastes bitter and slightly pungent. The H. moellendorffii medicinal material is shown in Fig. 3. Microscopic identification
Cross section: The cross section of H. moellendorffii is observed to have following microscopic identification features:
The phellem layer is a number of columns of cells, and the phelloderm is narrow. The cortex has a few oil chambers; and the phloem is broad, accounting for more than 1/2 of the entire root, and there are many oil chambers, radially 40-80 μm, tangentially 26-146 μm, surrounded by 6-10 secretory cells. The cambium forms a ring; and the xylem vessels are numerous, 25-52 μm in diameter, single or clustered. The rays are 1-2 rows of cells in width. The parenchyma cells contain starch granules. Fig. 4, Fig. 5, and Fig.6 show the details.
Powder: The powder is light yellow, and shows numerous starch granules. Single granules are quasi-circular, 5-14 μm in diameter, and some show umbilical points, dotted or herringbone. The layered patterns are not obvious, and there are many compound granules. Most of the vessels are reticulated, and 25-52 μm in diameter. The cork cells are brown-yellow. The vittae are mostly broken, and there are brown-yellow secretions in the secretory cells. Fibers are rare, 16-27 μm in diameter. Fig. 7 shows the details.
Thin layer identification
H. moellendorffii from 6 habitats in Guizhou was selected as the test samples. A certain amount of powder (2 g) was added with 10 ml of methanol, obtaining a mixture, which was ultrasonically treated for 15 min and filtered, giving the filtrate as a test solution. A certain amount of the control medicinal material of H. moellendorffii (2 g) was prepared into a control medicinal material solution by the same method. Then, 10 μl of each of the test solutions and the control medicinal material solution were transferred and dotted on the same silica gel G thin-layer plate and developed with petroleum ether (60-90 ℃)-ethyl acetate (7∶3) as the developing agent. After air-drying, the plate was inspected under UV light (365 nm), as shown in Fig. 8.
Determination of extract
H. moellendorffii root powder (Nwdh3) was taken and determined according to the extract determination method in Chinese Pharmacopoeia (2015 edition, general rule 2201). Using different concentrations of ethanol, it can be determined that when dilute ethanol was used as the solvent, the extract content of the Chinese medicinal material, H. moellendorffii roots was higher. The results are shown in Table 2.
Through the selection of the cold soaking method, hot soaking method and extraction solvent, it can be seen that when the hot soaking method used dilute ethanol as the extraction solvent, the content of H. moellendorffii extract was higher. It was tentatively decided to use dilute ethanol as the solvent, and the hot soaking method was used to determine H. moellendorffii extracts. The extracts of 10 batches of H. moellendorffii were determined (calculated in dry product). The results are shown in Table 3. According to the determination results, the extracts of 10 batches of samples were in the range of 22.22-35.51, with an average value of 26.9. It was tentatively decided that the extract standard should not be less than 22.0%.
Pengfei XIA et al. Study on the Quality Standard of Heracleum moellendorffii Hance.
S: Control medicinal material of H. moellendorffii; 1: Leishan County, Southeastern Guizhou; 2: Bayang Town, Ziyun County, Anshun City; 3: Jiucaiping, Bijie City; 4: purchased (Ziyun County, Anshun City); 5: Yangwu Town, Ziyun County, Anshun City; 6: purchased (Anshun City).
Conclusions and Discussion
The quality standards of H. moellendorffii were included in the 2003 edition of Quality Standards for Chinese Medicinal Materials and Ethnic Medicinal Materials in Guizhou Province, but the original standards are relatively simple, and have no detailed descriptions of traits and microscopic identification, and no extract and thin-layer identification experiments, so there are certain limitations in evaluating the quality of medicinal materials. Based on the original standards, in this study, we improved the character and microscopic identification, and added extract and thin-layer identification. Since furanocoumarins reported in the literatures are the main medicinal ingredients, and the National Institute for the Control of Pharmaceutical and Biological Products did not have relevant reference materials, no content determination method were established at this time. In TLC thin-layer identification, the control medicinal material and the samples were developed with different developing solvents including petroleum ether (60-90 ℃)-acetone (7∶3), petroleum ether (60-90 ℃)-ethyl acetate (7∶3) and ethyl acetate-methanol-water (5∶1∶0.5), and examined under 365 nm ultraviolet light, and it was found that with petroleum ether (60-90 ℃)-ethyl acetate (7∶3), the spots were clear in color, and the resolution and repeatability were good. This study can provide a scientific basis for the improvement of the 2003 version of Quality Standards for Chinese Medicinal Materials and Ethnic Medicinal Materials in Guizhou Province.
References
［1］ Editorial Board of Flora of China, Chinese Academy of Sciences. Flora of China［M］. Beijing: Science Press, 1974. (in Chinese)
［2］ SONG PS, ZHU JR, et al. Materia medica study of Chinese herbal medicines Heracleum produced in Gansu ［J］. Acta Chinese Medicine and Pharmacology, 2004, 32(4): 24, 25. (in Chinese)
［3］ Editorial Board of Quality standards for Chinese medicinal materials and ethnic medicinal materials in Guizhou Province. Quality standards for Chinese medicinal materials and ethnic medicinal materials in Guizhou Province［M］. Guiyang: Guizhou Science and Technology Publishing House, 2003. (in Chinese)
［4］ RAO GX, YANG Q, CAI F, et al. Chemical composition of Heracleum moellendorffii Hance.［J］. Journal of Yunnan University of Traditional Chinese Medicine, 1997, 17(3): 4-6. (in Chinese)
［5］ ZHOU LL, ZENG JG. Chemical constituent and pharmacological research progress of Heracleum hemsleyanum Diels［J］. Modern Chinese Medicine, 2019, 21(12): 1739-1748. (in Chinese)