Analysis and Study on Quality Control of Yao Medicine Damnacanthus macrophyllus Based on Methods in Chinese Pharmacopoeia

Jiangcun WEI, Xiumei MA, Yanxiu WANG, Bihua NONG, Chunli TANG*, Jingrong LU, Fengxian ZHAO

1. Guangxi International Zhuang Medicine Hospital, Nanning 530201, China; 2. The First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning 530023, China

Abstract [Objectives] To establish a quality analysis method for Yao medicine Damnacanthus macrophyllus Sieb. ex Miq. var. giganteus (Makino) Koidz..[D. indicus Gaertn. f. var. giganteus Makino]. [Methods] The moisture, total ash and alcohol extracts of D. macrophyllus medicinal materials were determined according to the experimental methods in the Chinese Pharmacopoeia (2020). [Results] All the 10 batches of D. macrophyllus medicinal materials from different collection times and different regions in Guangxi had the same plant morphological characteristics. The experimental results indicate that the moisture was 9.81%-11.52%, the total ash content was 7.37%-9.53%, the acid-insoluble ash content was 1.10%-1.97%, and the extract was 8.25%-10.22%. [Conclusions] This study provides a scientific basis for establishing a quality standard for Yao medicine D. macrophyllus medicinal materials, and it is expected to better utilize and develop D. macrophyllus.

Key words Damnacanthus macrophyllus Sieb. ex Miq. var. giganteus (Makino) Koidz..[D. indicus Gaertn. f. var. giganteus Makino], Moisture, Total ash content, Alcohol extract, Quality analysis

1 Introduction

Yao medicine Chuanlianzhu, also named Jijinshen, Huangjipang, Huangjilang, and Changye Shuzhugen, is the root ofDamnacanthusmacrophyllusSieb. ex Miq. var. giganteus (Makino) Koidz..[D.indicusGaertn. f. var. giganteus Makino], a plant of the genusDamnacanthusGaertn.fin family Rubiaceae. It is slightly bitter and sweet in taste, and neutral in nature. According to the records of theNationalAssemblyofChineseHerbalMedicine,D.macrophyllushas functions of invigorating qi and nourishing blood, and is astringent hemostatic, and mainly used for women with metrorrhagia, intestinal subwind blood syndrome, weakness and blood deficiency[1-2]. It is mainly distributed in Guangxi, Guangdong, Hunan, Guizhou, and Yunnan of China. At present, relevant literature reports that the main active components of Yao medicineD.macrophyllusare flavonoids, phenolic acids, anthraquinones, and iridoid glycosides. This herbal medicine is commonly used in the folk to replace Morindae Officinalis Radix for the treatment of weakness and blood deficiency[3-4].

Yao medicineD.macrophyllusis often used by the people of Guangxi to treat tumors and other diseases, but it has not been widely utilized and developed[5]. To useD.macrophyllusin a safe and effective manner, it is extremely important to study the quality standards of this medicinal material. However, there are currently few studies on the quality standards of Chinese medicinal materials in China. The applicants for the project have performed a pre-experimental study on the components of Yao medicineD.macrophyllus. Guangxi is the main production area and main clinical use area of Yao medicineD.macrophyllus. In order to developD.macrophyllusmedicinal resources, explore folk herbal resources, and establish a scientific quality control method for medicinal materials to determine the appropriate harvesting period, we have carried out a systematic research and systematic experiment on the characteristics ofD.macrophyllusmedicinal materials, various inspections of medicinal materials and determination of extracts,etc. The results are expected to provide a scientific basis for the development and utilization ofD.macrophyllusresources, and a sufficient basis for clinical safe and reasonable application.

2 Materials and methods

2.1 Materials

2.1.1Instruments and reagents. 101A-3E electric air blowing dryer and SX2-4-10 box type resistance furnace-muffle furnace (Shanghai Laboratory Instrument Works Co., Ltd.); SQP electric balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.); HWSZ6 electroheating thermostatic water bath (Shanghai Bluepard Instruments Co., Ltd.).

Ethanol and methanol (analytical reagent (AR), Sinopharm Group Chemical Reagent Co., Ltd.), water was pure water.

2.1.2Source of medicinal materials. A total of 10 batches ofD.macrophyllusmedicinal materials used in the experiment were collected from different regions in Guangxi and they were backing dried. The materials were identified by Zeng Chao, chief Chinese pharmacist from Department of Pharmacy, the First Affiliated Hospital of Guangxi University of Chinese Medicine, as the root ofDamnacanthusmacrophyllusSieb. ex Miq. var. giganteus (Makino) Koidz..[D.indicusGaertn. f. var. giganteus Makino]. The specific sources are listed in Table 1. AllD.macrophyllusmedicinal materials from different collection times and different regions have the same plant morphological characteristics (Fig.1).

Table 1 Information of samples of Damnacanthus macrophyllus

Note: the left is fresh material, and the right is dried material.

2.2 Methods

2.2.1Moisture measurement. With reference to the moisture determination method specified in General Rule 0832 of 2020ChinesePharmacopoeiaVolume 4, we measured the moisture. Firstly, weighed 3.0 g ofD.macrophylluspowder and screened through No.2 sieve, placed these materials dried to constant weight in flat weighing bottle, the thickness less than 5 mm, loosened the test samples less than 10 mm, precisely weighed, opened the bottle cap and dried at 105 ℃ for 5 h, covered the cap, moved to a dry container, cooled for 30 min, and make precision measurement, dried at the 105 ℃ for one hour, cooled down and weighed, till the difference of two consecutive weighing not higher than 5 mg. Finally, calculated the moisture of theD.macrophyllusmedicinal material according to the loss of moisture weight.

2.2.2Determination of total ashes. With reference to the ash determination method specified in General Rule 2302 of 2020ChinesePharmacopoeiaVolume 4,took about 4 g ofD.macrophyllusmedicinal powder, screened through No.2 sieve, placed in a crucible that was dried to constant weight, weighed and heated slowly, took care to avoid burning medicinal materials, and placed the crucible in the muffle furnace when it was completely carbonized. In the furnace, gradually increased the temperature to 600 ℃ to completely ash and reach the constant weight. Finally, calculated the content of total ashes of theD.macrophyllusmedicinal material according to the total ash weight of medicinal residues.

2.2.3Determination of acid insoluble ash. Took the ash obtained above, slowly added up approximately 10 mL of dilute hydrochloric acid to the crucible, covered the crucible with a watch glass, placed in a water bath and heated for 10 min, rinsed the watch glass with about 5 mL of hot water, merged the washing liquid into the crucible, filtered with ash-free filter paper, rinsed the residue in the crucible on the filter paper with pure water, and washed until the lotion does not show chloride reaction. Moved the filter paper and filter residue to the same crucible, weighed, and heated it slowly. When it was completely carbonized, placed the crucible in the muffle furnace, and gradually increased the temperature to 600 ℃ to completely ash and reach the constant weight. Finally, calculated the percentage of acid insoluble ashes of the medicinal materials according to the weight of the acid insoluble ash residue of the medicinal material.

2.2.4Measurement of extracts. The medicinal materials used for the determination need to be crushed to pass through the No.2 sieve and mix well. Used the pure water, 10% ethanol, 20% ethanol, 30% ethanol, 40% ethanol, 50% ethanol, 60% ethanol, 70% ethanol, 80% ethanol, and 95% ethanol as solvents, determined the extract ofD.macrophylluswith reference to the extract determination method (cold soaking method and hot soaking method with the pure water and ethanol as solvent) specified in General Rule 2201 acid soluble extract determination of 2020ChinesePharmacopoeia(Volume 4).

The extract was determined in accordance with the extract measurement method specified in General Rule 2201 of 2020ChinesePharmacopoeia(Volume 4). Precisely weighed 3.0 g of the medicinal material, placed in a 100-mL conical flask, accurately measured 40% ethanol 60 mL sealed the conical flask, weighed, let it stand for one hour, and refluxed for one hour. Cooled down, removed the conical flask, tightly sealed, made up the weight loss, shook well, filtered with a dry filter, accurately measured 25 mL of the filtrate, and placed it in an evaporating dish that has been dried to a constant weight. Dried for 3 h at 105 ℃, cooled in a drier for 30 min, and precisely and quickly weighed. Finally, calculated the percentage of extracts of theD.macrophyllusmedicinal material according to the weight of medicinal extract.

3 Results and analysis

3.1 MoistureFrom Table 2, it can be known that there are certain differences in the moisture of 10 batches ofD.macrophyllusmedicinal materials in different regions of Guangxi and the average moisture content was 10.62%. The moisture content ofD.macrophyllusmedicinal materials collected in Gongcheng County, Guangxi in November 2020 was the highest among the 10 batches ofD.macrophyllusmedicinal materials, which is 11.52%. Therefore, the limit was set at 120% of the highest value[6-8], and it is tentatively determined that the moisture content of Yao medicineD.macrophyllusis not higher than 14%.

Table 2 Determination results of moisture and ash content of Damnacanthus macrophyllus (n=10, %)

3.2 Measurement of ashesFrom Table 2, it can be seen that there are certain differences in the total ash and acid insoluble ash content of 10 batches ofD.macrophyllusmedicinal materials in Guangxi. Experimental results showed that the total ash content of medicinal materials was 7.37%-9.53%. The total ash content ofD.macrophyllusmedicinal materials collected in Fuchuan County, Guangxi in November 2020 was the highest in 10 batches ofD.macrophyllusmedicinal materials, which is 9.53%. Therefore, the limit was set at 120% of the highest value[6-8]. Thus, it is tentatively determined that the total ash content ofD.macrophyllusis not higher than 12.0%. Experimental results showed that the acid insoluble ash content ofD.macrophyllusmedicinal materials was 1.10%-1.97%. The highest content of acid insoluble ash was 1.97% collected in Pingle County, Guangxi in December 2020. Therefore, the limit was set at 120% of the highest value[6-8]. Thus, it is tentatively determined that the acid insoluble ash content ofD.macrophyllusis not higher than 3.0%.

3.3 ExtractAccording to the experimental results, in the cold soaking method and hot soaking method, the extract content ofD.macrophyllusmeasured with 30% ethanol as the solvent was the highest, so 30% ethanol was used as the extraction solvent (Table 3).

Table 3 Test of pure water, ethanol cold and hot soaking methods for the content of extracts in Damnacanthus macrophyllus

The 10 batches ofD.macrophyllusfrom different production areas and collection seasons in Guangxi were measured and the results are shown in Table 4.

From Table 4, it can be known that there are certain differences in the content of extracts of 10 batches ofD.macrophyllusmedicinal materials in different production areas of Guangxi. The experimental results indicate that the enthanol-soluble extract content was 8.25%-10.22% and the average content was 9.52%. The extract content ofD.macrophyllusmedicinal materials collected in Cenxi City of Guangxi in November 2020 was the highest among the 10 batches ofD.macrophyllusmedicinal materials, which is 8.25%. Therefore, the limit was set at 80% of the highest value[6-8], and it is tentatively determined that the extract content of Yao medicineD.macrophyllusis not higher than 7.0%.

Table 4 Determination results of content of ethanol-soluble extracts in Damnacanthus macrophyllus (n=13, %)

4 Conclusions

In this experiment, the moisture, ash and extract of 10 batches ofD.macrophyllusmedicinal materials from different production areas and collection seasons in Guangxi were investigated and determined. The results showed that the moisture ofD.macrophyllusmedicinal materials was not higher than 14.0%, and the total ash content was not higher than 12.0%, the insoluble ash content was not higher than 3.0%, and the extract was not lower than 7.0%. The results are compliant with the regulations, indicating that different production areas and collection times have an effect on the moisture, ash and extract content of the medicinal materials. These results are expected to provide a scientific basis for the establishment, utilization and development of the quality standards forD.macrophyllusmedicinal materials.