Jiangcun WEI, Jiabao MA, Jian CUI, Zhenying FU, Xianyi SHI, Yan MA, Lifu WEI, Zhengteng YANG*

1. Guangxi International Zhuang Medical Hospital, Nanning 530201, China; 2. The First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning 530023, China; 3. Guangxi Zhuang Yao Medicine Center of Engineering and Technology, Nanning 530200, China

Abstract [Objectives] To establish a method for quality analysis of Laggera alata. [Methods] The water content, total ash and alcohol-soluble extract of Laggera alata were determined according to the method of 2015 edition of Chinese Pharmacopoeia. [Results] Ten batches of Laggera alata from different producing areas and different collection time in Guangxi had the same plant morphology and medicinal properties. The experimental results were as follows: the water content was 5.19%-10.86%; the total ash content was 5.91%-10.74%; the acid-insoluble ash content was 0.44%-0.92%; the extract content was 14.64%-19.95%. [Conclusions] The experimental results can provide scientific basis for the development and utilization of Laggera alata and the establishment of its quality standard.

Key words Laggera alata, Character identification, Quality analysis

1 Introduction

Laggeraalata(D. Don) Sch. Bip. ex Oliv. belongs to the Composite family, and the whole herb can be used as medicine. It is mainly distributed in Guangxi, Yunnan, Sichuan and Guizhou[1], enriching the diversity of local biological species[2]. Its fresh branches and leaves give off a bad smell after being mashed, and it is used in clinic due to its anti-bacterial, anti-inflammatory, heat-clearing and detoxification properties, so it is called "stinky elixir"[3]. Many compound preparations based on it have also achieved good results in clinical application[4-5].L.alatais often used for a variety of diseases such as fall injury, burn, snake bite, cold and cough, rheumatoid arthritis, skin eczema, abdominal pain and diarrhea. At present, it is mainly used in the treatment of respiratory diseases[6], colds[7], viral infections[8]and other diseases, but its effective components are not clear. In recent years, the therapeutic effect ofL.alataplants has attracted more and more attention. The results showed thatL.alatamainly contained sesquiterpenes, flavonoids, phenolic acids and so on. Pharmacological experiments have proved thatL.alatahas the effects of treating acute and chronic inflammation, anti-tumor, anti-virus and anti-microbial activity.

L.alatais a common folk Chinese herbal medicine. At present, there is little research on its medicinal resources, and there is no relatively perfect quality standard control method. In order to develop the medicinal resources ofL.alata, establish a scientific quality standard control method of medicinal materials, and determine its suitable harvest time, a systematic experimental study on the characteristics, components and extract determination ofL.alatawas carried out in this paper.

Now there are some studies on the chemical constituents ofL.alatain China, but there are no related quality analysis reports. The medicinal materials ofL.alatawere studied systematically, and the results could provide a scientific basis for the development and utilization ofL.alataresources.

2 Instruments and reagents

2.1 Instruments101A-3E electric blast drying box (Shanghai Experimental Instrument Factory Co., Ltd.); SX2-4-10 box resistance furnace-muffle furnace (Shanghai Experimental Instrument Factory Co., Ltd.); HWSZ6 electric-heated thermostatic water bath (Shanghai Yiheng Scientific Instrument Co., Ltd.); pure water filter (Millipore, France); SQP electronic analytical balance (Sedorius Scientific Instruments Beijing Co., Ltd.).

2.2 ReagentsAlcohol (analytical pure, China Pharmaceutical Group Chemical Reagent Co., Ltd.).

3 Medicinal materials

A total of ten batches ofL.alataused in the experiment were collected from different areas of Guangxi. The collected part was whole grass and the drying method was drying in the sun. It was identified asL.alata(D. Don) Sch. Bip. ex Oliv. by the teacher of the Department of Traditional Chinese Medicine, School of Pharmacy, Guangxi University of Chinese Medicine. The specific sources are shown in Table 1.

4 Identification of plant morphology and characters

4.1 Morphological identificationL.alatais a perennial herb with a special smell. It tillers or sometimes does not tiller or rarely tillers. Its stem is stout, erect, about 50-120 cm tall. Its base is about 8 mm in diameter, and its base is woody. Its internodes are 1.0-2.5 cm long and 2-5 mm wide. The upper part has many branches and marks. Stems often form two small tips in broad places or at the ends. Leaves are alternate, oblong, sessile. Its base extends into 2-5 cm wide wings, with sparsely denticulate margin. Its midrib is stout and protruding on both sides. The capitulum is pendulous, 1 cm in diameter. As a raceme, it is born in the axils of winged branchlets and arranged into large raceme panicles in the shape of cylinders or spires at the top of the stem. The raceme is mostly arranged in panicles or axillary at the top of stems and branches. Its corolla is yellow, silky and about 7 mm long. In the center, there are few bisexual flowers, the corolla is tubular, and the flowers are 6-8 mm long (Fig.1).

Table 1 Sample information of Laggera alata

Fig.1 Medicinal herbs of Laggera alata (collected from De’e Township, Longlin County, Baise City in May 2019)

4.2 Characters of medicinal materialsThe stem ofL.alatais cylindrical, about 0.5-1.0 cm in diameter, and has 6 rows of stem wings. The surface of the tender stem is green to grayish green, with fine longitudinal lines, and the old stem is brown and lignified. The leaf blade is hard and brittle, wrinkled and broken, densely covered with fine and soft hair which is green to grayish green. It is hard and slightly tough, and the pith accounts for about two thirds of the diameter, whitish. The capitulum is grayish brown and has a special smell.

5 Quality analysis

5.1 Water content determination

5.1.1Determination method. The water content was determined according to the 0832 General Rule in the fourth edition ofChinesePharmacopoeiain 2015. 2.5 g of herb powder (sifted by 20-mesh sieve) was laid in a flat weighing bottle dried to constant weight, the thickness of which was less than 5 mm. The thickness of the loose sample was less than 10 mm and was accurately weighed. After drying the medicinal materials at 105 ℃ for 5 h and putting the cap on the bottle, it was moved to the dryer, cooled for 30 min, and weighed precisely. After drying at 105 ℃ for 1 hour, it was cooled and weighed until the difference in weighing twice in a row did not exceed 5 mg. According to the weight lost, the water content (%) in medicinal materials was calculated.

5.1.2Determination results. With reference to the determination method under Section5.1.1, the water content of ten batches ofL.alatafrom different producing areas and at different collection time in Guangxi was determined. The results are shown in Table 2 below.

Table 2 Determination results of water content in Laggera alata (n=10, %)

It can be seen from Table 2 that there were some differences in the water content of ten batches ofL.alatain Guangxi, with an average water content of 8.11%. The water content ofL.alatacollected in Binyang County, Guangxi in November 2018 was the highest in ten batches ofL.alata, reaching 10.86%. Therefore, with a limit of 120% of the maximum value[12], it was tentatively determined that the water content ofL.alatawas not higher than 13.03%.

5.2 Ash content determination

5.2.1Total ash. According to the 2302 Ash Determination Method in the fourth general rule of the 2015 edition ofChinesePharmacopoeia, 4 g of herb powder (sifted by 20-mesh sieve) was put into a crucible dried to constant weight, weighed (accurate to 0.01 g), and heated slowly. Attention should be paid to prevent the medicinal materials from burning. When they were completely carbonized, the crucible should be placed in the muffle furnace. The temperature gradually rose to 600 ℃, making it completely become ash and reach constant weight. According to the weight of residue, the content of total ash (%) in medicinal materials was calculated.

5.2.2Acid-insoluble ash. The ash obtained above was taken and slowly mixed with about 10 mL of diluted hydrochloric acid in the crucible. The crucible was covered with a surface dish and heated for 10 min in a water bath. The surface dish was washed with 5 mL of hot water, the washing solution was merged into the crucible and filtered with ashless filter paper. The residue in the crucible was washed on the filter paper with water and washed until the washing solution did not show chloride reaction. The filter paper and the filter residue were moved to the same crucible and weighed. After slow heating to complete carbonization, the crucible was put in the muffle furnace, and the temperature gradually rose to 600 ℃, making it completely become ash and reach constant weight. According to the weight of residue, the content of acid-insoluble ash (%) in medicinal materials was calculated.

5.2.3Determination results. With reference to the determination methods under Section5.2.1and Section5.2.2, ten batches ofL.alatafrom different producing areas and at different collection time in Guangxi were determined. The results are shown in Table 3 below.

Table 3 Determination results of ash content in Laggera alata (n=10, %)

It can be seen from Table 3 that there were some differences in the total ash and acid-insoluble ash content of ten batches ofL.alatain Guangxi. The total ash content ofL.alatacollected in Binyang County, Guangxi in November 2018 was the highest, reaching 10.74%, so the limit was set at 120% of the maximum value[12]. It was tentatively determined that the total ash content ofL.alatawas not higher than 12.89%. The acid-insoluble ash content ofL.alatacollected in Binyang County, Guangxi in November 2018 was the highest, reaching 0.92%, so the limit was set at 120% of the maximum value[12]. It was tentatively determined that the acid-insoluble ash content ofL.alatawas not higher than 1.10%.

5.3 Determination of extractThe medicinal materials used for determination should be crushed so that they could be sifted by 20-mesh sieve and mixed evenly.

5.3.1Selection of determination conditions. Pure water, 10% alcohol, 25% alcohol, 45% alcohol, 65% alcohol, 85% alcohol and 95% alcohol were used as solvents forL.alata, respectively. It was determined according to the 2201 Alcohol-soluble Extract Determination Method in the fourth general rule of the 2015 edition ofChinesePharmacopoeia(cold-maceration method and hot-maceration method, using pure water and alcohol as solvent). The content of extract in medicinal materials was determined, and the results are shown in Table 4.

The experimental results showed that the content of extract fromL.alatadetermined by 10% alcohol solvent hot-maceration method was the highest in cold-maceration method and hot-maceration method, so 10% alcohol was used as extraction solvent.

Table 4 Investigation of pure water and alcohol cold-maceration method and hot-maceration method (%)

5.3.2Determination method. It was determined according to the 2201 Alcohol-soluble Extract Determination Method in the fourth general rule of the 2015 edition ofChinesePharmacopoeia. 2.5 g of medicinal material was precisely weighed and placed in the 100 mL conical bottle. 60 mL of water was precisely measured and weighed after it was stoppered. After standing for 1 h, it was refluxed for 1 h. After cooling, the conical bottle was taken, stoppered, shaken well, and filtered with a drying filter. 25 mL of filtrate was placed in an evaporation dish that was dried to a constant weight. After steam drying in water bath, it was dried at 105 ℃ for 3 h, cooled in a dryer for 30 min, and weighed quickly and accurately. According to the weight of the extract, the content (%) of the extract inL.alatawas calculated.

5.3.3Determination results. With reference to the determination method under Section5.3.2, ten batches ofL.alatafrom different producing areas and at different collection time in Guangxi were determined. The results are shown in Table 5.

Table 5 Determination results of the content of extract in Laggera alata (n=10, %)

As can be seen from Table 5, there were some differences in the extract content of ten batches ofL.alatain Guangxi, with an average content of 17.38%. The extract content ofL.alatacollected in Pingle County, Guilin City in May 2019 was the lowest, reaching 14.64%, so the limit was set at 80% of the lowest value[13]. It was tentatively determined that the extract content ofL.alatawas not less than 11.71%.

6 Conclusion and discussion

In this experiment, the water, ash and extract contents ofL.alatafrom different producing areas and at different collection time in Guangxi were determined. The experimental results were as follows: the water content ofL.alatawas not more than 13.03%; the total ash content was not more than 12.89%; the acid-insoluble ash content was not more than 1.10%; the extract content was not less than 11.71%. All the testing results were in line with the regulations. The results showed that the water, ash and extract contents of medicinal materials were affected by different producing areas and collection time. The experimental results can provide a scientific basis for the development and utilization ofL.alataand the establishment of the quality standard of medicinal materials.