Evaluation of Hydroxycinnamic Acid Content, Antioxidant Activity of Siberian crab Apple (Malus baccata (L.) Borkh) Leaves

Nesterova N. V.¹, Teplov I.S.², Bokov D.O.², Sergunova E.V.², Luferov A.N.²,

Samylina I.A.², Zhevlakova A.К.², Savvateev A.M², Nesterov G.V²

¹Peoples Friendship University, of Russia 117198, Moscow Miklukho-Maklaya str.6,

²Sechenov First Moscow State Medical University, 8 Trubetskaya St.,

bldg. 2, Moscow, 119991, Russian Federation

*Corresponding Author E-mail: nestero-nadezhda@yandex.ru

ABSTRACT:

Objective: Given the significant interest of researchers in studying a wide range of pharmacological effects characteristic of apple raw materials due to the presence of various groups of biologically active substances (BAS), the object of the study was the Siberian crab apple leaves (Malus baccata (l.) Borkh), growing in the Botanical Garden of Moscow State University. Lomonosov, as well as in gardens and parks of ecologically safe areas of the Moscow region, including from varietal plants “Street Parade” (Archiland plant nursery) harvested during the flowering, fruiting and autumn period of fruit fall (September 2024). Materials and methods. The analysis was performed on a GILSON high-performance liquid chromatograph, model 305 (France), manual injector, model RHEODYNE 7125 USA. A metal column with a size of 4.6 x 250 mm KROMASIL C18 was used as the stationary phase, and a mixture of acetonitrile – water – phosphoric acid concentrated in a ratio of 20:80: 0.05 was used as the mobile phase. The quantitative content of hydroxycinnamic acids in the M. baccata leaves was determined by spectrophotometry in terms of caffeic acid (3,4-dioxycinnamic acid, 3-(3,4-dihydrophenyl)-2-propenic acid). Results and discussions. The authors studied the composition of the polyphenolic fraction, isolated a number of hydroxycinnamic acids and carried out their quantitative assessment. Based on the analysis of data from HPLC analysis of phenolic derivatives, caffeic, gallic, chicory, chlorogenic, neochlorogenic, cinnamic acids, catechin, epicatechin, vicenin, rutin, coumarin, epicatechingallate quercitin, floretin, floridzine, apigenin were determined. The presence of flavonoids floretin, floridzine and vicenin, previously found in extracts from the leaves of forest and domestic apple trees, can be considered as a probable identifier of the genus Malus. Hydroxycoric acids such as caffeic, gallic, chicoric, chlorogenic, neochlorogenic, and cinnamic acids have been identified. The content of hydroxycinnamic acids in terms of caffeic acid, depending on the phase of vegetation, was revealed by spectrophotometric analysis. The authors found that the maximum amount of hydroxycinnamic acids accumulates in the plant during the fruiting phase. Conclusion. The analysis allows us to determine and identify phenolic derivatives in the fraction in extracts from apple leaves, which will simplify the standardization of raw materials in the preparation of appropriate regulatory documentation

KEYWORDS: M. baccata tree, Hydroxycinnamic acids, HPLC, Spectrophotometry.

INTRODUCTION:

Siberian crab apple (Malus baccata (L.) Borkh.) is a woody plant belonging to the Malus genus (Rosaceae family), widely distributed in Eastern Siberia, Transbaikalia, and the Far East. It is successfully cultivated in the European part of the Russian Federation. It is a deciduous shrub up to 5 m high or a small tree no more than 10 m high. It occurs in the undergrowth of mixed and deciduous forests, along the edges, and can be found scattered in floodplain forests. The leaves and fruits of the M. baccata tree, along with the M. baccata, have long been used in medicine of different peoples as an anti-schadenfreude agent for colds, lung diseases, anemia, and were recommended as a general tonic and tonic. It was believed that the fruits of the M. baccata prevent the development of obesity, hypertension, gout and chronic rheumatism.

Recently, raw materials – fruits and leaves of apple trees of various types-have again begun to attract the attention of researchers, due to the presence of a wide range of biologically active substances of various groups^1-3, as well as various pharmacological effects, including antioxidant, anti-inflammatory, and radioprotective activity.^4-9 Using chromatographic methods, the presence of hydroxycinnamic ketones and flavonoids in the fruits and leaves of forest and domestic apple trees was established. For raw materials - leaves and fruits of the M. baccata tree, the composition of organic acids is determined, represented by oxalic, fumaric, malic, citric, acetic, benzoic, sorbic and ascorbic acids of sugars, among which fructose, glucose and sucrose are determined, as well as trace elements.¹⁰ The authors found that the M. baccata leaves accumulate a greater number of macro-and microelements than in fruits.¹¹At the same time, a comprehensive phytochemical analysis of the M. baccata leaves, which forms significant commercial areas in the Russian Federation, and is also effectively cultivated, was not carried out. All the above indicates that the study of raw materials of apple berry is a promising and urgent problem, the solution of which will expand the range of medicinal products. vegetable raw materials.

The aim of the work is to study the composition and quantitative determination of hydroxycinnamic acids in the leaves of the M. baccata leaves in different phases of vegetation.

Materials and methods:

The object of our research was the M. baccata leaves harvested from cultivated plants growing in the Botanical Garden of the Lomonosov Moscow State University, as well as in gardens and parks of ecologically safe areas of the Moscow region, including the varietal plants “Street Parade” (Archiland plant nursery), harvested during the flowering, fruiting and autumn periods. period of fruit fall (September 2024). Raw materials were dried by the method of shadow drying with stirring.

The qualitative composition of biologically active substances was studied in extracts from apple leaves obtained by extraction with 70% ethyl alcohol, using the HPLC method, which is widely used in the analysis of various phenolic compounds, including hydroxycinnamic acids.^12,13 The analysis was performed on a high-performance liquid chromatograph manufactured by GILSON, model 305 (France), manual injector, model RHEODYNE 7125 USA с, followed by computer processing of the study results using the Multichrome program for Windows. 4.6 x 250 mm KROMASIL C18 metal column with a particle size of 5 microns was used as the stationary phase. A mixture of acetonitrile – water – phosphoric acid concentrated in the ratio 20: 80: 0.05 was used as the mobile phase. The analysis was performed at room temperature. The eluent feed rate is 1.0 ml/min. The analysis duration is from 40 to 60 minutes. Detection was performed using a GILSON» UV/ VIS model 151 UV detector at a wavelength of 370 nm.

The quantitative content of hydroxycinnamic acids in the M. baccata leaves was determined by spectrophotometry and in terms of caffeic acid (3,4-dioxycinnamic acid, 3 - (3,4-dihydrophenyl) - 2-propenic acid). Preliminary sample preparation of raw materials included grinding to the size of particles passing through anito filter with a hole diameter of 2 mm. Crushed raw materials weighing 2 g (weighing the exact weight was carried out on a Mettler Toledo ME 204 analytical balance) were placed in a flask with a capacity of 200 ml and 70 ml of distilled water was added. The flask was connected to a return refrigerator and heated in a water bath for 15 minutes. Extraction was repeated. The resulting extracts were cooled at room temperature and filtered through a paper filter. The extracts were quantitatively transferred to a 200 ml volumetric flask and the volume of the distilled water solution was adjusted to the mark. Then, 1 ml of the previously obtained extract was transferred to a 50 ml volumetric flask and the volume of the solution was adjusted to the mark using 20% ethyl alcohol. The optical density of the resulting solution was measured at a wavelength of 325 nm, since this value is analytical for caffeic acid. 20% ethyl alcohol was used as the reference solution.

Theratio of the amount of hydroxycinnamic acids (X, %) in the leaves of a M. baccata tree in terms of caffeic acid was calculated by the formula:

where Ax is the optical density of the test solution; is the specific absorption index of caffeic acid at 325 nm, which is 782; m – weight of the suspension of crushed M. baccata leaves; – aliquot volume, ml; W – humidity, %.

The antioxidant activity of M. baccata leaves was evaluated by spectrophotometry using a technique based on the interaction of compounds that have antioxidant activity with the 1,1-diphenyl-2-picryl-hydrozyl radical (DPPH), which has an absorption maximum at a wavelength of 517 nm. During the ongoing reaction of DPPH with substances with an antioxidant effect, a decrease in the number of free radicals occurs, which leads to a decrease in absorption.

2.5 g (exact weight) of dried and crushed apple berry leaves were placed in a 100 ml flask, 70% ethanol was added and heated in a boiling water bath with a return refrigerator for an hour. The resulting extract was cooled to room temperature and filtered through a paper filter into a 25 ml volumetric flask, if necessary, bringing the volume to the mark with 70% ethanol.

К 2 мл A DPPH solution (Sigma-Aldrich, USA) with a concentration of 0.5 mM and a volume of 2 ml (solution in 96% ethanol) was added to 2 ml of the test extraction and the optical density of the resulting mixture was determined on an Analytik Jena spectrophotometer at a wavelength of 517 nm with a cuvette layer thickness of 10 mm every 15 minutes for 1 hour. The degree of DPPH radical inhibition was determined by the formula:

% inhibition = *100%,

where is the adsorption value of the control solution, is the adsorption value after the reaction after a fixed time.

The control solution was prepared by mixing 2 ml of DPPH solution with a concentration of 0.5 mM and 2 ml of 96% ethanol.

Statistical processing of the results of the study was carried out in accordance with the requirements of GPM 42-0111-09 “Statistical processing of the results of a chemical experiment”.

RESULTS AND DISCUSSION:

Chromatographic analysis of ethanol extraction from the leaves of a M. baccata harvested in different phases of vegetation showed the identity of biologically active substances, the composition of which is presented in Table 1 and Figure 1. During the study, chromatographic profiles containing 29 peaks were obtained for all samples, among which, by comparing the retention times of the components with the retention times of standard samples, caffeic, gallic, chicoric, chlorogenic, non-chlorogenic, cinnamic acids, catechin, epicatechin, vicenin, rutin, coumarin, EGCG, quercitin, floretin, floridzine, apigenin.

The results of quantitative determination of hydroxycinnamic acids in terms of caffeic acid for raw materials of different preparation times are presented in the tables.

Figure 1: HPLC chromatogram of the alcohol extract from the M. baccata leaves. Compounds names: 1- caffeic acid, 2- gallic acid, 3 - chicory acid, 4 - catechin, 5 - epicatechin, 6 - chlorogenic acid, 7 - vicenin, 8 - neochlorogenic acid, 9 - rutin, 10 - coumarin, 11- epicatechingallate, 12 - quercetin, 13 - phloretin, 14 - phlorizin, 15 - cinnamic, 16 - acid, 17 - apigenin.

Table 1: Results of the study of phenolic compounds in dry and fresh raw materials M. baccata leaves, by HPLC.

No.	Time, min	Area,%	Compound name	Structural formula
1	4.46	83.72	Caffeic acid
2	5.32	61.36	Gallic acid
3	5.64	2073.48	Chicory acid
4	7.15	1643.69	Catechin
5	7.69	2034.61	Epicatechin
6	8.53	342.65	Chlorogenic acid
7	9.07	682.60	Vicenin
8	9.61	141.42	Neochlorogenic acid
9	10.22	948.18	Rutin
10	11.85	85.44	Coumarin
11	12.61	84.98	Epicatechingallate
12	17.88	219.72	Quercetin
13	18.32	91.47	Phloretin
14	21.24	266.55	Phlorizin
15	23.76	17.74	Сinnamic acid
16	24.47	6.84	Apigenin

Table 2: Results of quantitative determination of hydroxycinnamic acids in the M. baccata leaves in terms of caffeic acid during the flowering period

Sample No	Sample name	Hydroxycinnamic acid content in terms of caffeic acid, %
1	M. baccata leaves collected at the Lomonosov Moscow State University	2.28±0.61
2	M. baccata leaves collected in the park area of Istra, Moscow region	2.42±0.74
3	M. baccata leaves collected at the “Rassvet” garden cooperative	2.35±0.92
4	M. baccata leaves of the “Street Parade” variety (Archiland plant nursery)	2.41±0.72

Table 2: Results of quantitative determination of hydroxycinnamic acids in the M. baccata leaves in terms of caffeic acid during the fruiting period

Sample No	Sample name	Hydroxycinnamic acid content in terms of caffeic acid, %
1	M. baccata leaves collected at the Lomonosov Moscow State University	3.19±0.48
2	M. baccata leaves collected in the park area of Istra, Moscow region	3.39±0.75
3	M. baccata leaves collected at the “Rassvet” garden cooperative	3.25±0.54
4	M. baccata leaves of the “Street Parade” variety (Archiland plant nursery)	3.41±0.73

Table 3: Results of quantitative determination of hydroxycinnamic acids in apple leaves in terms of caffeic acid in the autumn period of tree fall and harvest

Sample No	Sample name	Hydroxycinnamic acid content in terms of caffeic acid, %
1	M. baccata leaves collected at the Lomonosov Moscow State University	2.21±0.69
2	M. baccata leaves collected in the park area of Istra, Moscow region	2.39±0.81
3	M. baccata leaves collected at the “Rassvet” garden cooperative	2.12±0.77
4	M. baccata leaves of the “Street Parade” variety (Archiland plant nursery)	2.34±0.61

Statistical processing of data from five parallel measurements showed that the total content of hydroxycinnamic acids in the M. baccata leaves in terms of caffeic acid reached the maximum value for all the studied samples during the beginning of fruiting, which is 28.7% more than the corresponding indicators during the flowering period and 30.7% - during the fruit fall (autumn period) for the sample collected in the Botanical Garden of the Lomonosov Moscow State University.

To study the antioxidant activity of raw materials by spectrophotometry, we carried out sample preparation to obtain extracts from the leaves яof berry yarrow in a ratio of 1:10 using 70% ethyl alcohol as an extractant.¹⁴

The measurements were carried out in the range of absorption values of 0.2-0.7. The experiment was repeated 3 times, the confidence interval was 0.95 using the student’s coefficient. The results are shown in table 5.

Table 5: Results of studying the antioxidant activity of the M. baccata leaves

Time, min*	Degree of DPPH radical inhibition (%) in extraction from the leaves collected at different phases
Time, min*	flowering phase	beginning of fruiting	fall and fruit collection phase
15	34.56 ± 0.83	37.62 ± 0.83	32.76± 0.96
30	38.19 ± 0.83	41.41 ± 1.04	36.81± 0.88
45	43.28 ± 0.83	46.63 ± 1.35	41.43± 1.21
60	44.79 ± 0.83	47.39 ± 1.32	42.27± 1.19

Note: *Time elapsed after the start of the reaction between the DPPH solution and the extraction from the M. baccata leaves

According to the results shown in Table 5, it can be concluded that the antioxidant activity of the M. baccata leaves.^15-19 DPPH radical inhibition occurs in the first 15 minutes and then gradually decreases over the course of an hour.^20,21

CONCLUSION:

Based on the analysis of HPLC data of phenol derivatives, caffeic, gallic, chicoric, chlorogenic, non-chlorogenic, cinnamic acids, catechin, epicatechin, vicenin, rutin, coumarin, EGCG, quercitin, floretin, floridzine, and apigenin were determined. The presence of flavonoids phloretin, floridzin, and vicenin, previously detected in extracts from the leaves of forest and domestic apple trees, can be considered as a probable identifier of the Malus genus.

The class of hydroxycinnamic acids found in the M. baccata leaves includes caffeic, gallic, chicory, chlorogenic, non-chlorogenic, and cinnamic acids. The content of hydroxycinnamic acids in terms of caffeic acid was determined by spectrophotometric analysis depending on the vegetation phase. It is established that the maximum amount of hydroxycinnamic acids accumulates in the plant during the fruiting phase. The presence of antioxidant activity in the M. baccata leaves was established, and for the first 15 minutes the degree of inhibition was 32.76-37.62%, and after an hour after mixing extracts from the leaves of the apple tree with a solution of the DPPH radical was in the range of 42.27-47.39%.

CONFLICTS OF INTEREST:

None.

ACKNOWLEDGEMENTS:

This research has been supported by the RUDN University Strategic Academic Leadership program grant number 033323-2-000.

References:

1. Dadwal V, Joshi R, Gupta M. Comparative metabolomics of Himalayan crab apple (Malus baccata) with commercially utilized apple (Malus domestica) using UHPLC-QTOF-IMS coupled with multivariate analysis. Food Chemistry. 2023; 402: 134529. doi: 10.1016/j.foodchem.2022.134529

2. Kishor A, Attri BL, Brijwal M, Kumar A, Narayan R, Singh D, Debnath S, Mer MS, Tiwari VK. Physico-chemical characterization of wild apple (Malus baccata) in Kumaon hills of Uttarakhand. Ecology. Environment and Conservation. 2016; 22: 287-91.

3. Kumar C, Singh SK, Pramanick KK, Verma MK, Srivastav M, Singh R., Bharadwaj C, Naga K.C. Morphological and biochemical diversity among the Malus species including indigenous Himalayan wild apples. Scientia Horticulturae. 2018; 233: 204-219. doi: 10.1016/j.scienta.2018.01.037

4. Petkova NT, Ognyanov M, Inyutin B, Zhelev P, Denev P. Phytochemical composition and antioxidant activity of Malus baccata (L.) Borkh. fruits. Food Science and Applied Biotechnology. 2020; 3(1): 47-55. doi: 10.30721/fsab2020.v3.i1.46

5. Dadwal V, Agrawal H, Sonkhla K, Joshi R, Gupta M. Characterization of phenolics, amino acids, fatty acids and antioxidant activity in pulp and seeds of high altitude Himalayan crab apple fruits (Malus baccata). J Food Sci Technol. 2018; 55(6): 2160-2169, doi: 10.1007/s13197-018-3133-y

6. Song C, Chen H, Kim SA, Lee JS, Choung ES, Zhang Z, Kim SY, Kim JH, Cho JY. Anti-Inflammatory Functions of Methanol Extract from Malus baccata (L.) Borkh. Leaves and Shoots by Targeting the NF-κB Pathway. Plants (Basel). 2022; 11(5): 646. doi: 10.3390/plants11050646

7. Wang L, Li X, Wang Z. Whole body radioprotective effect of phenolic extracts from the fruits of Malus baccata (Linn.) Borkh. Food Funct. 2016; 7(2): 975-81. doi: 10.1039/c5fo01377a

8. Han M, Li A, Shen T, Meng J, Lei Y, Zhang X, Liu P, Gan L, Ao L, Li H. Phenolic compounds present in fruit extracts of Malus spp. show antioxidative and pro-apoptotic effects on human gastric cancer cell lines. J Food Biochem. 2019; 43(11): e13028. doi: 10.1111/jfbc.13028

9. Liu Y, Zhao Y, Zhuo Y, Li Y, Meng J, Wang Y, Li H. Ultrasound-Assisted Extraction of Anthocyanins from Malus 'Royalty' Fruits: Optimization, Separation, and Antitumor Activity. Molecules. 2022; 4; 27(13): 4299. doi: 10.3390/molecules27134299

10. Bakhanova V, Antsupova TP. Chemical composition of the fruit of the M. baccata tree (Malus baccata (L.) Borkh.) in the conditions of Buryatia. Izvestiya Samarskogo nauchnogo tsentra Rossiiskoi akademii nauk. 2017; 2 (3): 416-419.

11. Bahanova MV, Chuparina EV, Batueva YuM, Lovtsova NM, Novolodskaya AA. Specificity of the elemental composition of the M. baccata tree in natural and urbanized conditions of the Republic of Buryatia. Bulletin of the Buryat State University. Biology. Geografiya. 2018; 3: 72-74.

12. Lenchyk LV, Saidov NB. Investigation of phenolic compounds in cherry leaves extract. Research Journal of Pharmacy and Technology. 2017; 10(12): 4402-4406. doi: 10.5958/0974-360X.2017.00811.3

13. Lenchyk LV, Ovezgeldiyev D, Shapoval OV, Baiurka SV, Ayaou A. Study of chemical composition and diuretic activity of cherry fruit extract. Research J. Pharm. and Tech. 2018; 11(7): 3036-42, doi: 10.5958/0974-360X.2018.00559.0

14. Jeba RC, Sandhya GA, Das N, Shau CS, Kumar SA. Anticancer activity of microwave assisted polyphenolic compounds extracted from combinations of Curcuma longa and Camellia sinensis against lung cancer cell line. Research Journal of Pharmacy and Technology. 2023; 16(5): 2192-6. doi: 10.52711/0974-360X.2023.00360

15. Mitra P, Dutta AK, Mandal NC, Acharya K. Phytochemical study and antioxidative property of polyphenol rich fraction from Termitomyces medius. Research J. Pharm. and Tech. 2019; 12(9): 4287-4294. doi: 10.5958/0974-360X.2019.00737.6

16. Rangasamy P, Hansiya VS, Maheswari PU, Suman T, Geetha N. Phytochemical analysis and evaluation of in vitro antioxidant and anti-urolithiatic potential of various fractions of Clitoria ternatea L. blue flowered leaves. Asian J. Pharm. Ana. 2019; 9(2): 67-76. doi: 10.5958/2231-5675.2019.00014.0

17. Abriyani E, Fikayuniar L. Screening phytochemical, antioxidant activity and vitamin C assay from bungo perak-perak (Begonia versicolar Irmsch) leaves. Asian J. Pharm. Res. 2020; 10(3): 183-187. doi: 10.5958/2231-5691.2020.00032.5

18. Madhu C, Swapna J, Neelima K, Shah MV. A Comparative evaluation of the antioxidant activity of some medicinal plants popularly used in India. Asian Journal of Research in Pharmaceutical Science. 2014; 2(3): 98-100

19. Kalidas S, Kameswari B, Devi P, Madhumitha B, Meera R, Merlin NJ. Phyto-physico chemical evaluation, antioxidant activities and diuretic activity of leaves of Lagerstroemia reginae. Asian J. Research Chem. 2008; 1(2): 83-87

20. Zeroual S, Daoud I, Gaouaoui R, Ghalem S. In vitro and molecular docking studies of DPPH with Phoenix dactylifera L. (Deglet-Nour) crude fruits extracts and evaluation of their antioxidant activity. Asian J. Research Chem. 2020; 13(1): 52-59. doi: 10.5958/0974-4150.2020.00012.7

21. Habchi A, Dekmouche M, Hamia C, Saidi M, Yousfi M, Bouguerra A. Extraction of Phenolic Compounds of six Algerian date (Phoenix dactylifera L.) cultivars from Ain-Saleh region, using Reflux method and Screening of Antioxidant Activity in vitro. Asian Journal of Research in Chemistry. 2021; 14(3): 161-7. doi: 10.52711/0974-4150.2021.00030.

Received on 13.12.2024 Revised on 05.07.2025

Accepted on 08.10.2025 Published on 20.05.2026

Available online from May 25, 2026

Research J. Pharmacy and Technology. 2026;19(5):2367-2373.

DOI: 10.52711/0974-360X.2026.00339

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

n	f				P, %	t (P, f)
M. baccata leaves collected in the flowering phase
5	4	2.276	0.000125	0.0112	95	2.78	0.01392	0.61
M. baccata leaves collected in the fruiting phase
5	4	3.193	0.000150	0.0122	95	2.78	0.01523	0.48
M. baccata leaves collected in the fruit fall phase
5	4	2.213	0.000153	0.01237	95	2.78	0.01538	0.69