Research Article

Quantification of Vitamin C Content in Rosa brunonii Lindl Rosehips: A Potential Vitamin Supplement

Salim Saifullah, Muhammad Ilyas, Bashir Ullah and Sanam Zarif Satti*

Biological Sciences Research Division, Pakistan Forest Institute, Peshawar, Khyber Pakhtunkhwa, Pakistan.

Received | October 21, 2022; Accepted | December 16, 2022; Published | December 26, 2022

*Correspondence | Sanam Zarif Satti, Biological Sciences Research Division, Pakistan Forest Institute, Peshawar, Khyber Pakhtunkhwa, Pakistan; Email: sanamzarifsatti@gmail.com

Citation | Saifullah, S., Ilyas, M., Ullah, B. and Satti, S.Z., 2022. Quantification of vitamin C content in Rosa brunonii Lindl rosehips: A potential vitamin supplement. Pakistan Journal of Forestry, 72(2): 69-75.

DOI | https://dx.doi.org/10.17582/journal.PJF/2022/72.2.69.75

Keywords | Vitamin C, Rosa brunonii L., Rosehip, Vitamin supplement

Copyright: 2022 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Vitamin C is an antioxidant that is soluble in water and primarily functions in a protective capacity. The antioxidant properties of vitamin C enable it to effectively scavenge free radicals, thereby protecting lipids, proteins, and DNA from oxidative damage (EFSA Panel on Dietetic Products et al., 2017). It also helps plants fight against oxidative stress caused by biotic or abiotic stressors due to its antioxidant capabilities and excellent redox regeneration mechanism (Gest et al., 2013). The significance of vitamin C in being able to resist various stressors is emphasized by the upregulation of biosynthetic and recycling enzymes in response to unfavorable environmental circumstances (Gallie, 2013; Höller et al., 2015). It reduces iron in the active site of several dioxygenases to Fe2+ and post-translationally modifies procollagen to produce collagen. It is a cofactor for several enzymes that are responsible for the hydroxylation of proline and lysine, which is essential for the formation of the stable structure of collagen (Mandl et al., 2009; Pekkala et al., 2003; Peterkofsky, 1991). It helps hydroxylase synthesize carnitine and norepinephrine (Dunn et al., 1984; Rebouche, 1991). Vitamin C also plays a significant role in various hydroxylation reactions that are dependent on cytochrome P450. These reactions include the conversion of cholesterol into bile acids, the breakdown of exogenous substances like drugs and pollutants, and the production of steroid hormones (Kobayashi et al., 2014).

Vitamin C is categorized as a vitamin solely for selected vertebrate species, like Homo sapiens, due to their inability to endogenously produce it. The human incapacity to produce vitamin C is attributed to the functional deficiency of the gene encoding l-gulonolactone oxidase, which is the final enzyme in the animal biosynthesis of this particular vitamin (Linster and Van Schaftingen, 2007). Ascorbate plays a crucial role in the biosynthesis of collagen and carnitine in animals, which are essential components of blood vessels, tendons, ligaments, scar tissue, and skin (Levine, 1986; Vissers et al., 2020). The significance of vitamin C lies in its crucial role in the maintenance and repair of teeth, bones, and cartilage, as well as its involvement in the process of wound healing. In recent years, there has been significant interest in enhancing the nutritional value of plants by increasing their vitamin C content, given that plant-based foods are the primary source of vitamin C in human diets (Chen et al., 2003; Hancock and Viola, 2005; Naqvi et al., 2009). According to some research, vitamin C’s antioxidant properties make it effective against cancer, cardiovascular disease, and atherosclerosis (Harris, 2013).

Rosa brunonii pertains to the botanical family Rosaceae. This particular plant species holds significant importance in the field of medicine. It is a spindly climbing shrub that can reach a height of 5-6 meters. Its scented flowers, which typically bloom from April to June, are characterized by white petals. R. brunonii is found growing as a wild shrub in various regions of Pakistan, including Gilgit, Kashmir, Poonch, Hazara, Swat, Chitral, Kurram, Murree, and Balouchistan (Ishaque et al., 2021). It has been utilized in traditional medicine to address a range of health issues. The bark infusions are employed as a means of purifying the blood, while the leaf extract is applied externally to promote wound healing when mixed with pure water. Additionally, the tea of the flowers is utilized to alleviate diarrhea and constipation, and the flower extract is employed to address skin and eye ailments. The local inhabitants of the Galliyat region in Pakistan have also been known to use the fruit of R. brunonii to treat skin diseases (Amjad et al., 2017; Bano et al., 2014; Ishaque et al., 2017; Khan et al., 2015). This paper will examine and evaluate the content of vitamin C in Rosa brunonii rosehips from various Murree locations focusing its potential as vitamin supplement. Rose hips are a nutritious and versatile addition to the diet. They have been consumed for centuries due to their health benefits and culinary uses. Quantifying vitamin C in rose hips for vitamin supplements is crucial for ensuring accuracy, product consistency, health benefits, and consumer confidence. It helps both manufacturers and consumers make informed decisions about the use of rose hip supplements in their diets.

Materials and Methods

Materials and reagents

Plant material (rosehips of Rosa brunonii) were collected from five localities of Murree hills i.e., Ghora Gali (GG), Danoi (DA), Kotli Sattian (KS) Bhurban (BH), and Lower Topa (LT) at an elevation ranging from 1000 to 2000 meters. Chemical reagents including ammonium molybdate, oxalic acid, Sulphuric acid, EDTA, metaphosphoric acid, acetic acid, and L-ascorbic acid were purchased from Sigma Aldrich and used as received. All the solvents used in this study were HPLC grade or distilled before use.

Sampling and physical examination of rosehips

Three sets of twenty rosehips prepared by randomly picking up from each sample collected from five different localities of Muree Hills. Each set of twenty rosehips were assessed to measure average fruit weight, flesh ratio, fruit length, and fruit width of rosehips.

Vitamin C extraction

To extract vitamin C from rosehips, first an oxalic acid-EDTA solution was prepared by dissolving 6.3 g of oxalic acid and 744 mg of EDTA in 1000 mL distilled water (Bajaj and Kaur, 1981). Subsequently, 10 g of each sample were blended in 100 mL oxalic acid-EDTA solution to prepare homogenized suspension through electric blender. The resultant suspension was filtered to remove the rosehips residue and large solid particles. The filtrate was collected and then centrifuge at 3000 rmp to remove any micron level particles. The supernatant was transferred to amber reagent bottle, properly labeled, and stored for farther analysis.

Devolvement of calibration curve

A stock solution of vitamin C with a concentration of 0.1% (m/V) was prepared through the dissolution of 100 mg of L-ascorbic acid in a 100 mL solution of oxalic acid-EDTA. To construct the calibration curve, discrete volumes of 1.0, 2.0, 3.0, 4.0, 5.0, and 6.0 mL were transferred into individual volumetric flasks that were pre-filled with a 50 mL solution of oxalic acid-EDTA. To each flask, an additional 5 mL of metaphosphoric acid-acetic acid solution was added, followed by 10 mL of a 10% (v/v) sulfuric acid solution, and finally 20 mL of ammonium molybdate solution. The solution obtained was subjected to dilution using distilled water until a final volume of 250 mL was achieved. Subsequently, the absorbance of the solution was measured at 760 nm using spectrophotometric techniques. The solution of oxalic acid and EDTA was prepared through the dissolution of 6.3 g and 744 g of oxalic acid and EDTA in 1000 mL of distilled water, respectively. A solution of metaphosphoric acid and acetic acid was produced through the dissolution of 15 g of metaphosphoric acid in 40 mL of acetic acid, followed by the addition of distilled water to achieve a final volume of 500 mL. The preparation of an ammonium molybdate solution involved the dissolution of 5 g of ammonium molybdate in 100 mL of distilled water.

Quantification of vitamin C

To quantify the vitamin C in rosehips samples, all the vitamin C extraction samples stored in amber reagent bottle were shake well and then 50 mL extraction of each sample was transferred into individual volumetric flasks. To each flask, an additional 5 mL of metaphosphoric acid-acetic acid solution was added, followed by 10 mL of a 10% (v/v) sulfuric acid solution, and finally 20 mL of ammonium molybdate solution. The solution obtained was subjected to dilution using distilled water until a final volume of 250 mL was achieved. Subsequently, the absorbance of the solution was measured at 760 nm using spectrophotometric technique.

Results and Discussion

Physical examination of rosehips

The physical properties of the rosehips collected from five different localities are shown in Table 1. The weights and dimensions of representative rosehips were recorded as minimum (Min), maximum (Max), and mean values (Average). The mean weights of the rosehips exhibited a range between 4.54 and 7.92 g. The results indicate that LT exhibited the greatest mean weight of 7.92 g, followed by DA (7.25 g). BH and KS had almost similar average weight of rosehips whereas GG had the lowest wight among all samples. Average rosehip flesh ratio varied from 62.35% to 77.13%. The weight of a rosehip is an important factor in determining its nutritional value, as larger rosehips generally contain more nutrients. The flesh ratio, which refers to the ratio of pulp to seeds within the rosehip, is also important, as the pulp contains more nutrients than the seeds. Generally, rosehips with a higher flesh ratio are considered to be of higher nutritional value. Average fruit width was ranging from 6.45 to 9.65 mm. Average fruit length was found in the range of 12.15 to 16.93 mm. The length and width of rosehips can also affect their nutritional value. Larger rosehips may contain more flesh and seeds, which can contribute to their overall nutritional content. However, it is important to note that other factors, such as the variety of rosehip and the conditions in which it was grown, can also impact its nutritional content (Ercişli and Eşitken, 2004; Lee and Kader, 2000).

 

Table 1: Rosehip weight, rosehip flesh ratio, rosehip length, and rosehip width of Rosa Brunonii collected from five different localities of Muree Hills i.e., Ghora Gali (GG), Danoi (DA), Kotli Sattian (KS) Bhurban (BH), and Lower Topa (LT).

Samples	Rosehip weight (g)			Rosehip flesh ratio (%)			Rosehip width (mm)			Rosehip length (mm)
	Min	Max	Average	Min	Max	Average	Min	Max	Average	Min	Max	Average
GG	4.23	4.64	4.54 ±0.15	56.34	65.27	62.35 ±0.25	5.78	7.15	6.45 ±0.23	9.67	14.45	12.15 ±0.45
DA	5.95	7.84	7.25 ±0.12	67.58	78.94	75.18 ±0.28	7.74	9.12	8.63 ±0.25	11.86	18.24	15.84 ±0.23
KS	5.86	7.32	6.18 ±0.17	65.73	68.25	67.36 ±0.35	6.45	8.38	7.35 ±0.15	10.38	16.85	14.15 ±0.16
BH	5.75	7.96	6.35 ±0.15	66.86	71.49	69.75 ±0.18	6.36	8.96	7.57 ±0.25	12.72	17.18	14.75 ±0.43
LT	6.67	8.32	7.92 ±0.14	72.92	84.35	77.13 ±0.23	8.58	10.45	9.65 ±0.32	13.65	19.63	16.93 ±0.45

 

Devolvement of calibration curve

Several techniques have already been reported for determining vitamin C. The spectrophotometric or calorimetric methods are commonly employed, depending on the reactions with phenylhydrazinium chloride, dimethoxyquinone, 4-nitrobenxenediazonium fluoroborate, 2, 4-dinitrophenylhydrazine, diazotized 4-methoxy-2-nitroaniline, and 2, 6-dichloroindophenol (Besada, 1987; Kelly and Latzko, 1980; Khadka and Pathak, 2023; Soliman, 1979). The molybdophosphate complex has also been identified as a means of determining vitamin C content (Vishnikin et al., 2011). The initial trials indicated that the response depends on the proportion of sulfuric acid to vitamin C. The stability of the blue color generated persists for approximately 20 hours at room temperature. The spectral absorption peak occurs at a wavelength of 760 nm, and the linear relationship between the concentration of the solution and its absorbance, defined by Beer’s law, is valid within the concentration range of 2–25 µg/mL (Figure 1).

 

 

Quantification of vitamin C

Vitamin C content, total soluble solids (TSS), and total dry weight (TDW) of the rosehips collected from five different localities are shown in Figure 2. The average vitamin C content varied from 918 to 2985 mg/100 g. Whereas the TSS and TDW varied from 31 to 34% and 34 to 40%, respectively. BH had the highest vitamin C content with 2985 mg/100 g along with 36% TSS and 40% TDW, followed by GG, which had 2535 mg/100 g vitamin C content along with 35% TSS and 39% TDW. KS and LT had almost similar vitamin C content, whereas DA had the lowest weight among all samples. Typically, fresh consumption of rosehip fruits is not a common practice. The fruits undergo processing procedures to yield various consumable products such as soft drinks, syrup, marmalade, jellies, and jams (Ahmad and Anwar, 2016). Thus, the desirable and significant properties of rosehips include higher levels of vitamin C content, TSS, and TDW.

There are several factors affecting the content of vitamin C in wild rosehips. The stage of maturity of the rosehips affects the vitamin C content. Generally, rosehips that are fully ripe contain more vitamin C than those that are not yet fully mature (Medveckienė et al., 2021). The harvesting time can also affect the vitamin C content (Kallio et al., 2002). Rosehips that are harvested in the late fall or early winter generally have a higher vitamin C content than those harvested earlier in the season. The processing method used to prepare the rosehips affects the vitamin C content. Heat, light, and air all cause vitamin C to break down, so processing methods that minimize exposure to these factors help preserve the vitamin C content in rosehips (Santos and Silva, 2008). The storage conditions of the rosehips affected the vitamin C content. Storing rosehips in a cool, dark, and dry place also helps preserve the vitamin C content (Franke et al., 2004).

Conclusions and Recommendations

In summary, this study was conducted to quantify the vitamin C content in rosehips collected from various locations in the Murree Hills. The vitamin C content of the samples ranged from 918 to 2985 mg/100 g, with BH and GG having the highest vitamin C content. The vitamin C content of rosehips is affected by a variety of factors, so it’s important to take these factors into consideration when harvesting, processing, and storing the rosehips. The results concluded that rosehip from Rosa Brunonii is a rich source of vitamin C, and samples collected from various locations had a significant content of vitamin. Rosehips from these locations could be used as a potential vitamin supplement in producing syrup, marmalade, jellies, and jams.

Acknowledgement

The authors are grateful to the Pakistan Forest Institute, Peshawar for generously supporting this study.

Novelty Statement

Rose hips are a nutritious and versatile addition to the diet. Quantifying vitamin C in rose hips for vitamin supplements is crucial for ensuring accuracy, product consistency, health benefits, and consumer confidence.

Author’s Contribution

S. Saifullah: Conceptualization, Methodology, Data curation, Data interpretation, Data visualization, and writing first draft and writing revised drafts. M. Ilyas: Methodology, Data curation, and writing first draft. B. Ullah: Methodology, Data curation, and writing first draft. S. Z. Satti: Conceptualization, Supervision, Plant material collection, Reviewing and Editing original draft and revision.

Conflict of interest

The authors have declared no conflict of interest.

References

Ahmad, N. and Anwar, F., 2016. Rose hip (Rosa canina L.) oils. In: Essential oils in food preservation, flavor and safety. Elsevier. pp. 667-675. https://doi.org/10.1016/B978-0-12-416641-7.00076-6

Amjad, M.S., Qaeem, M.F., Ahmad, I., Khan, S.U., Chaudhari, S.K., Zahid, M.N., Shaheen, H. and Khan, A.M., 2017. Descriptive study of plant resources in the context of the ethnomedicinal relevance of indigenous flora: A case study from Toli Peer National Park, Azad Jammu and Kashmir, Pakistan. PLoS One, 12(2): e0171896. https://doi.org/10.1371/journal.pone.0171896

Bajaj, K. and Kaur, G., 1981. Spectrophotometric determination of L-ascorbic acid in vegetables and fruits. Analyst, 106(1258): 117-120. https://doi.org/10.1039/an9810600117

Bano, A., Ahmad, M., Hadda, T.B., Saboor, A., Sultana, S., Zafar, M., Khan, M.P.Z., Arshad, M. and Ashraf, M.A., 2014. Quantitative ethnomedicinal study of plants used in the skardu valley at high altitude of Karakoram-Himalayan range, Pakistan. J. Ethnobiol. Ethnomed., 10(1): 1-18. https://doi.org/10.1186/1746-4269-10-43

Besada, A., 1987. A facile and sensitive spectrophotometric determination of ascorbic acid. Talanta, 34(8): 731-732. https://doi.org/10.1016/0039-9140(87)80229-1

Chen, Z., Young, T.E., Ling, J., Chang, S.C. and Gallie, D.R., 2003. Increasing vitamin C content of plants through enhanced ascorbate recycling. Proc. Natl. Acad. Sci., 100(6): 3525-3530. https://doi.org/10.1073/pnas.0635176100

Dunn, W., Rettura, G., Seifter, E. and Englard, S., 1984. Carnitine biosynthesis from gamma-butyrobetaine and from exogenous protein-bound 6-N-trimethyl-L-lysine by the perfused guinea pig liver. Effect of ascorbate deficiency on the in situ activity of gamma-butyrobetaine hydroxylase. J. Biol. Chem., 259(17): 10764-10770. https://doi.org/10.1016/S0021-9258(18)90577-6

EFSA Panel on Dietetic Products, N., Allergies, Turck, D., Bresson, J.L., Burlingame, B., Dean, T., Fairweather-Tait, S., Heinonen, M., Hirsch-Ernst, K.I., Mangelsdorf, I. and McArdle, H.J., 2017. Vitamin C and protection of DNA, proteins and lipids from oxidative damage: Evaluation of a health claim pursuant to Article 14 of Regulation (EC) No 1924/2006. EFSA J., 15(1): e04685. https://doi.org/10.2903/j.efsa.2016.4588

Ercişli, S. and Eşitken, A., 2004. Fruit characteristics of native rose hip (Rosa spp.) selections from the Erzurum province of Turkey. N. Z. J. Crop Hortic. Sci., 32(1): 51-53. https://doi.org/10.1080/01140671.2004.9514279

Franke, A.A., Custer, L.J., Arakaki, C. and Murphy, S.P., 2004. Vitamin C and flavonoid levels of fruits and vegetables consumed in Hawaii. J. Food Composit. Anal., 17(1): 1-35. https://doi.org/10.1016/S0889-1575(03)00066-8

Gallie, D.R., 2013. The role of L-ascorbic acid recycling in responding to environmental stress and in promoting plant growth. J. Exp. Bot., 64(2): 433-443. https://doi.org/10.1093/jxb/ers330

Gest, N., Gautier, H. and Stevens, R., 2013. Ascorbate as seen through plant evolution: The rise of a successful molecule? J. Exp. Bot., 64(1): 33-53. https://doi.org/10.1093/jxb/ers297

Hancock, R.D. and Viola, R., 2005. Improving the nutritional value of crops through enhancement of L-ascorbic acid (vitamin C) content: Rationale and biotechnological opportunities. J. Agric. Food chem., 53(13): 5248-5257. https://doi.org/10.1021/jf0503863

Harris, J.R., 2013. Subcellular biochemistry: Ascorbic acid: Biochemistry and biomedical cell biology (Vol. 25). Springer Science and Business Media.

Höller, S., Ueda, Y., Wu, L., Wang, Y., Hajirezaei, M.R., Ghaffari, M.R., von Wirén, N. and Frei, M., 2015. Ascorbate biosynthesis and its involvement in stress tolerance and plant development in rice (Oryza sativa L.). Plant Mol. Biol., 88: 545-560. https://doi.org/10.1007/s11103-015-0341-y

Ishaque, M., Bibi, Y. and Qayyum, A., 2021. Fruits of Rosa brunonii Lindle: An ethnomedicinal plant from foothills of Himalaya with antibacterial, antitumor and cytotoxic properties. Arab. J. Sci. Eng., 47: 155–163 https://doi.org/10.1007/s13369-021-05581-z

Ishaque, M., BiBi, Y., Valant–Vetschera, K.M., Schinnerl, J. and Bacher, M., 2017. Fruits of Rosa brunonii–a source of antioxidant phenolic compounds. Nat. Prod. Commun., 12(11): 1934578X1701201106. https://doi.org/10.1177/1934578X1701201106

Kallio, H., Yang, B. and Peippo, P., 2002. Effects of different origins and harvesting time on vitamin C, tocopherols, and tocotrienols in sea buckthorn (Hippophaë rhamnoides) berries. J. Agric. Food Chem., 50(21): 6136-6142. https://doi.org/10.1021/jf020421v

Kelly, G.J. and Latzko, E., 1980. Prospect of a specific enzymic assay for ascorbic acid (vitamin C). J. Agric. Food Chem., 28(6): 1320-1321. https://doi.org/10.1021/jf60232a051

Khadka, D. and Pathak, K., 2023. Spectrophotometric determination of total vitamin C content in different fruits and vegetables consumed in Tansen, Palpa. Tribhuvan J., 1(1): 51-57. https://doi.org/10.3126/tribj.v1i1.53497

Khan, M.P.Z., Ahmad, M., Zafar, M., Sultana, S., Ali, M.I. and Sun, H., 2015. Ethnomedicinal uses of edible wild fruits (EWFs) in Swat Valley, Northern Pakistan. J. Ethnopharmacol., 173: 191-203. https://doi.org/10.1016/j.jep.2015.07.029

Kobayashi, M., Hoshinaga, Y., Miura, N., Tokuda, Y., Shigeoka, S., Murai, A. and Horio, F., 2014. Ascorbic acid deficiency decreases hepatic cytochrome P-450, especially CYP2B1/2B2, and simultaneously induces heme oxygenase-1 gene expression in scurvy-prone ODS rats. Biosci. Biotechnol. Biochem., 78(6): 1060-1066. https://doi.org/10.1080/09168451.2014.910105

Lee, S.K. and Kader, A.A., 2000. Preharvest and postharvest factors influencing vitamin C content of horticultural crops. Postharv. Biol. Technol., 20(3): 207-220. https://doi.org/10.1016/S0925-5214(00)00133-2

Levine, M., 1986. New concepts in the biology and biochemistry of ascorbic acid. New Eng. J. Med., 314(14): 892-902. https://doi.org/10.1056/NEJM198604033141407

Linster, C.L. and Van Schaftingen, E., 2007. Vitamin C: Biosynthesis, recycling and degradation in mammals. FEBS J., 274(1): 1-22. https://doi.org/10.1111/j.1742-4658.2006.05607.x

Mandl, J., Szarka, A. and Banhegyi, G., 2009. Vitamin C: Update on physiology and pharmacology. Br. J. Pharmacol., 157(7): 1097-1110. https://doi.org/10.1111/j.1476-5381.2009.00282.x

Medveckienė, B., Kulaitienė, J., Levickienė, D. and Hallmann, E., 2021. The effect of ripening stages on the accumulation of carotenoids, polyphenols and vitamin C in rosehip species/cultivars. Appl. Sci., 11(15): 6761. https://doi.org/10.3390/app11156761

Naqvi, S., Zhu, C., Farre, G., Ramessar, K., Bassie, L., Breitenbach, J., Perez Conesa, D., Ros, G., Sandmann, G. and Capell, T., 2009. Transgenic multivitamin corn through biofortification of endosperm with three vitamins representing three distinct metabolic pathways. Proc. Natl. Acad. Sci., 106(19): 7762-7767. https://doi.org/10.1073/pnas.0901412106

Pekkala, M., Hieta, R., Kursula, P., Kivirikko, K.I., Wierenga, R.K. and Myllyharju, J., 2003. Crystallization of the proline-rich-peptide binding domain of human type I collagen prolyl 4-hydroxylase. Acta Crystallogr. D Biol. Crystallogr., 59(5): 940-942. https://doi.org/10.1107/S0907444903005420

Peterkofsky, B., 1991. Ascorbate requirement for hydroxylation and secretion of procollagen: Relationship to inhibition of collagen synthesis in scurvy. Am. J. Clin. Nutr., 54(6): 1135S-1140S. https://doi.org/10.1093/ajcn/54.6.1135s

Rebouche, C.J., 1991. Ascorbic acid and carnitine biosynthesis. Am. J. Clin. Nutr., 54(6): 1147S-1152S. https://doi.org/10.1093/ajcn/54.6.1147s

Santos, P. and Silva, M., 2008. Retention of vitamin C in drying processes of fruits and vegetables. A review. Dry. Technol., 26(12): 1421-1437. https://doi.org/10.1080/07373930802458911

Soliman, R., 1979. A sensitive colorimetric method for estimation of ascorbic acid. Talanta, 26(12): 1164-1166. https://doi.org/10.1016/0039-9140(79)80033-8

Vishnikin, A.B., Sklenářová, H., Solich, P., Petrushina, G.A. and Tsiganok, L.P., 2011. Determination of ascorbic acid with Wells-Dawson type molybdophosphate in sequential injection system. Anal. Lett., 44(1-3): 514-527. https://doi.org/10.1080/00032719.2010.500789

Vissers, M.C., Pullar, J.M. and Smirnoff, N., 2020. Chemistry and biochemistry of vitamin C in mammalian systems. In vitamin C, CRC Press. pp. 25-37. https://doi.org/10.1201/9780429442025-2