Preliminary Study on Quality Analysis of Yao Medicine Pileostegia tomentella Hand.-Mazz.

Xiaodong HUANG, Xiumei MA, Yong TAN, Jiangcun WEI*, Wen ZHONG*, Bing QING, Qingmei DENG, Hong LEI

1. Guangxi University of Chinese Medicine, Nanning 530200, China; 2. Guangxi International Zhuang Medicine Hospital Affiliated to Guangxi University of Chinese Medicine, Nanning 530201, China

Abstract [Objectives] To establish a method for quality analysis of Medicine Pileostegia tomentella Hand.-Mazz. [Methods] According to the testing method of Chinese Pharmacopoeia (2020 Edition), the water content, total ash and alcohol-soluble extract of P. tomentella Hand.-Mazz. were determined. [Results] 10 batches of P. tomentella Hand.-Mazz. from different habitats and in different collection time in Guangxi all had the same plant morphology and medicinal properties. The experimental results showed that the water content was 7.92%-10.35%, the total ash content was 5.53%-6.62%, the acid-insoluble ash content was 0.59%-0.76%, and the extract content was 11.63%-16.12%. [Conclusions] The identification method in this study can be used for the quality analysis of P. tomentella Hand.-Mazz., and the experimental results can provide a scientific basis for the development and utilization of P. tomentella Hand.-Mazz. and the establishment of its quality standard.

Key words Pileostegia tomentella Hand.-Mazz., Character identification, Quality analysis

1 Introduction

PileostegiatomentellaHand.-Mazz., a Yao medicine, is the vine and stem ofPileostegiaHook.f.et Thoms. in the Saxifragaceae family, and it is mainly distributed in Guangxi, Guangdong and other places. The whole plant has the effect of promoting blood circulation and dispelling blood stasis[1-2].P.tomentellaHand.-Mazz. has the effects of dispelling wind and dampness, removing blood stasis and relieving pain, and setting bones. It is often used clinically for treating soreness of waist and legs, rheumatism, numbness, and it is externally applied for treating traumatic injury, fracture, traumatic bleeding,etc.[1-2]It is recorded inChineseMateriaMedica[3],GuangxiMedicinalPlantsList[4],GuangxiFlora[5]and other books.P.tomentellaHand.-Mazz. has anti-tumor effect and can be used to treat a variety of malignant tumors, so it is called Zhongliuteng, also known as Xiao-liuteng[6]. Studies have shown thatP.tomentellaHand.-Mazz. has significant anti-tumor effect[7]. It is reported in the literature that the main effective components ofP.tomentellaHand.-Mazz. are flavonoids[7-8], coumarins[9-10], iridoids[10]and so on.P.tomentellaHand.-Mazz., a Yao medicine, is a commonly used Chinese herbal medicine. At present, there is little research on its medicinal resources, and there is no perfect quality standard control method. In order to develop the medicinal resources ofP.tomentellaHand.-Mazz. and establish a scientific quality control method of medicinal materials, the characteristics, tests and extracts ofP.tomentellaHand.-Mazz. were systematically studied in this paper.

2 Materials

2.1 Instruments and reagents101A-3E electric air blast drying oven (Shanghai Experimental Instrument Factory Co., Ltd.); SX2-4-10 box resistance furnace-muffle furnace (Shanghai Experimental Instrument Factory Co., Ltd.); HWSZ6 electric thermostatic water bath (Shanghai Yiheng Scientific Instrument Co., Ltd.); SQP electronic analytical balance[Sartorius Scientific Instrument (Beijing) Co., Ltd.]. Ethanol (analytically pure, Sinopharm Chemical Reagent Co., Ltd.).

2.2 Medicinal materialsA total of 10 batches of medicinal materials used in the experiment were collected from different areas in Guangxi, and were identified asP.tomentellaHand.-Mazz. by Professor Tan Yong from College of Pharmacy of Guangxi University of Chinese Medicine, which was the vine and stem of P. Hook.f.et Thoms. in the Saxifragaceae family. See Table 1 for specific sources.

3 Methods and results

3.1 Identification of plant morphology and traitsIt is evergreen climbing shrub, 16 m long; twigs, leaves and inflorescences were densely covered with light brown or rust-colored stellate pilose, often with 3-6 radial lines; old branches were cylindrical, subglabrous, grayish brown. Leaves were leathery, oblong or obovate-oblong, sparsely oblanceolate, 5-10 (18) cm long and 2.5-5.0 (8) cm wide. Apex was acute or broadly acute, with pointed end protruding. The base was round or slightly concave near petiole and heart-shaped, and the top two leaves of sparse branchlets were broadly cuneate. Edge was subentire or subapically triangular coarsely toothed or irregularly undulate. Young leaves were abaxially curly, sparsely covered with stellate hairs, later glabrescent, grayish green or yellowish green when dry. Its lower part was densely hairy, with 8-13 hairs on each side of leaf veins; petiole was 1.2-1.5 cm long.

Corymbose panicles were terminal, 10-25 cm long and wide; bracts were linear or subulate, 5-10 mm long, 1-2 mm wide, stellate hairy; flowers were white; pedicel was 2 mm long; calyx was tube cup-shaped, 2 mm high, and lobes were triangular, sparsely stellate hairy; petals were ovate, 2 mm long, caducous, glabrous; there were 8-10 stamens, filaments were 5-6 mm long; style was 1.5 mm long, stigma was paniculate, 4-6-lobed, hairy. Capsule was turbinate, flat-topped, 4 mm in diameter. It was sparsely stellate hairy, with persistent style and stigma, angulate, dark brown; seeds were small, 2 mm long, brown. The flowering period was from March to August, and the fruiting period was from September to December.

The root of this product was cylindrical, with yellowish brown to grayish brown surface, longitudinal wrinkles and marks, 0.2-2.0 cm in diameter; it was hard and difficult to break, with brown skin of the section and grayish white wood. The stem was cylindrical, with yellowish brown to grayish brown surface, longitudinal wrinkles and small marks. There were obvious irregular grooves in the cork of old stems, with a diameter of 0.6-3.0 cm. It was hard and difficult to break. The skin of the section was brown, the wood was gray-white, with fine radial texture. The pith was grayish yellow, or hollow. It had a slight smell and tasted slightly bitter.

3.2 Water content determination

3.2.1Determination method. According to the water content determination method in General Rule 0832, Volume IV ofChinesePharmacopoeia(2020Edition), 2.5 g of medicinal powder (sifted by a 24-mesh sieve) was laid flat in a flat weighing bottle dried to a constant weight, with a thickness of less than 5 mm and a thickness of loose test sample of less than 10 mm, which was accurately weighed. After drying medicinal material at 105 ℃ for 5 h, and the bottle cap was closed, the medicinal material was moved to the dryer, cooled 30 min, and weighed precisely. It was dried at 105 ℃ for 1 h, cooled and weighed until the difference between the weights measured twice in a row did not exceed 5 mg. According to the weight loss, the water content (%) in medicinal materials was calculated.

3.2.2Water content determination results. According to the determination method in Section3.2.1, the water content of 10 batches ofP.tomentellaHand.-Mazz. from different producing areas in Guangxi and at different collection time was determined, and the results are shown in Table 2.

From Table 2, it can be seen that the water content of 10 batches ofP.tomentellaHand.-Mazz. in Guangxi was different, and the average water content was 9.29%. In October, 2022, the water content ofP.tomentellaHand.-Mazz. collected in Hezhou City, Guangxi Zhuang Autonomous Region was the highest among 10 batches of medicinal materials, reaching 10.35%, so the limit was set at 120% of the highest value[11-14], and it was tentatively determined that the water content ofP.tomentellaHand.-Mazz., a Yao medicine in Guangxi, was not higher than 12.42%.

Table 2 Determination results of water content in Pileostegia tomentella Hand.-Mazz. (n=10, %)

3.3 Ash determination

3.3.1Determination of total ash. According to the ash determination method in General Rule 2302, Volume IV ofChinesePharmacopoeia(2020Edition), 4 g of medicinal powder (sifted by a 24-mesh sieve) was taken into a crucible dried to a constant weight, and weighed (accurate to 0.01 g). It was slowly heated to avoid burning the medicinal materials. When it was completely carbonized, the crucible was placed in a muffle furnace, and the temperature was gradually raised to 600 ℃, so that it completely turned into ashes and reached a constant weight. According to the weight of residue, the content of total ash (%) in medicinal materials was calculated.

3.3.2Determination of acid-insoluble ash. The ash obtained above was taken, and about 10 mL of dilute hydrochloric acid was slowly added to the crucible. The crucible was covered with a surface dish and heated in a water bath for 10 min. The surface dish was washed with 5 mL of hot water, and the washing liquid was transferred to the crucible and filtered with ashless filter paper. The residue in the crucible was washed on the filter paper with water until the washing solution did not show chloride reaction. The filter paper and the filter residue were moved to the same crucible and weighed. It was slowly heated, and when it was completely carbonized, the crucible was placed in a muffle furnace, and the temperature was gradually raised to 600 ℃, so that it completely turned into ashes and reached a constant weight. According to the weight of residue, the content of acid-insoluble ash (%) in medicinal materials was calculated.

3.3.3Ash determination results. According to the determination methods in Section3.3.1and3.3.2, the ash content of 10 batches ofP.tomentellaHand.-Mazz. from different producing areas in Guangxi and at different collection time were determined. The results are shown in Table 3.

Table 3 Determination results of ash content in Pileostegia tomentella Hand.-Mazz. (n=10, %)

It can be seen from Table 3 that there were some differences in the total ash and acid-insoluble ash content of 10 batches ofP.tomentellaHand.-Mazz. in Guangxi. The highest content of total ash was 6.62% forP.tomentellaHand.-Mazz. collected in Hezhou City, Guangxi in October 2022, so the limit was set at 120% of the highest value[7-10], and the total ash content ofP.tomentellaHand.-Mazz. was tentatively determined to be not higher than 7.94%. In the determination of acid-insoluble ash, the highest content was 0.76% forP.tomentellaHand.-Mazz. collected in Gongcheng County, Guilin City in October 2022, so the limit was set at 120% of the highest value[11-14], and the acid-insoluble ash ofP.tomentellaHand.-Mazz. was tentatively determined to be not higher than 0.91%.

3.4 Determination of extractsThe medicinal materials used for determination need to be crushed so that they can be sifted by a 24-mesh sieve and mixed evenly.

3.4.1Selection of determination conditions. 10% ethanol, 30% ethanol, 50% ethanol, 70% ethanol and 90% ethanol were used as solvents forP.tomentellaHand.-Mazz produced in Jingxi City, Guangxi, and the content of extracts in the medicinal materials was determined according to the alcohol-soluble extract determination method (cold-dipping method and hot-dipping method, using ethanol as solvent) in General Rule 2201, Volume IV ofChinesePharmacopoeia(2020Edition).

The results showed that the extract content ofP.tomentellaHand.-Mazz. determined by 70% ethanol solvent (hot-dipping method) was the highest, so 70% ethanol was used as the extraction solvent (Table 4).

Table 4 Investigation of ethanol cold-dipping method and hot-dipping method (%)

3.4.2Determination method. According to the determination method of alcohol-soluble extracts in General Rule 2201, Volume IV ofChinesePharmacopoeia(2020Edition), 2.5 g of medicinal materials were accurately weighed, placed in a 100 mL conical flask, 60 mL of water was accurately measured, weighed, and refluxed for 1 h after standing for 1 h. After cooling, the conical flask was taken, corked, and it was replenished with ethanol, shaken, and filtered with a drying filter. 25 mL of filtrate was precisely taken and placed in an evaporation dish dried to a constant weight. After evaporation in water bath, it was dried at 105 ℃ for 3 h, cooled in a dryer to cool for 30 min, and weighed quickly and accurately. According to the weight of the extract, the content of the extract (%) fromP.tomentellaHand.-Mazz. was calculated.

3.4.3Extract determination results. According to the determination method in Section3.4.2, 10 batches ofP.tomentellaHand.-Mazz. from different producing areas in Guangxi and at different collection time were determined, and the results are shown in Table 5.

From Table 5, it can be seen that there were some differences in the extract content of 10 batches ofP.tomentellaHand.-Mazz. in Guangxi, with an average content of 14.66%. In October, 2022, the extract content ofP.tomentellaHand.-Mazz. collected in Gongcheng County, Guilin City was the lowest in the determination of 10 batches of medicinal material extracts, reaching 11.63%, so the limit was set at 80% of the lowest value[11-14], and the extract ofP.tomentellaHand.-Mazz. was tentatively determined to be not less than 9.30%.

Table 5 Determination results of extract content in Pileostegia tomentella Hand.-Mazz. (n=10, %)

4 Conclusion and discussion

In this experiment, the water, ash and extract content ofP.tomentellaHand.-Mazz. from different producing areas in Guangxi and at different collection time was determined. The test results are as follows: forP.tomentellaHand.-Mazz., the water content was not more than 12.42%, the total ash content was not more than 7.94%, the acid-insoluble ash content was not more than 0.91%, and the extract content was not less than 9.30%. The test results were in accordance with the regulations. The results showed that different producing areas and different collection time had influence on the water content, ash and extract of medicinal materials. In this experiment, the water content, ash content and alcohol-soluble extract content ofP.tomentellaHand.-Mazz. were determined, and the experimental results can provide a scientific basis for the development and utilization ofP.tomentellaHand.-Mazz. and the formulation of its quality standards.