Phytochemical Constituents and Pharmacological Activities of Calendula officinalis Linn (Asteraceae): A Review

Calendula officinalis Linn. (Asteraceae) is used medicinally in Europe, China and India amongst several places in the world. It is also known as “African marigold” and has been a subject of several chemical and pharmacological studies. It is used in traditional medicine, especially for wound healing, jaundice, blood purification, and as an antispasmodic. Chemical studies have underlined the presence of various classes of compounds, the main being triterpenoids, flavonoids, coumarines, quinones, volatile oil, carotenoids and amino acids. The extract of this plant as well as pure compounds isolated from it, have been demonstrated to possess multiple pharmacological activities such as anti-HIV, cytotoxic, anti-inflammatory, hepatoprotective, spasmolytic and spasmogenic, amongst others. In this review, we have explored the phytochemistry and pharmacological activities of C. officinalis in order to collate existing information on this plant as well as highlight its multi-activity properties as a medicinal agent. This is as a result of the worldwide cultivation of the plant and increasing published reports on it.


INTRODUCTION
India is called the botanical garden of the world for its rich natural resources.Over 6,000 plants in India are used in traditional, folklore and herbal medicine [1].The Indian system of medicine has identified 1500 medicinal plants of which 500 are commonly used [1].Calendula officinalis Linn. is used medicinally in Europe, China, US and India.It belongs to the family, Asteraceae, and is commonly known as Zergul (Hindi), African marigold, Calendula, Common Marigold, Garden Marigold, Marigold, Pot Marigold (English), Butterblume (German), Chin Chan Ts'ao (Chinese), Galbinele (Romanian) and Ringblomma (Swedish) [2,3].

Taxonomic description
The plant is classified as shown in Table 1.

Habitat
The plant is native to Central and Southern Europe, Western Asia and the US [5].

Botanical description Flower and fruit
On the tip of each stem, there is a 5 to 7 cm composite flower head, as shown in Fig 1, consisting of an epicalyx of numerous narrow-lanceolate sepals, which are densely covered on both sides with glandular hairs.The inner section of the flower head is made up of orange-yellow tubular florets.The disc florets are pseudohermaphrodites but the female is sterile.The zygomorphic ray florets at the edge are female, their stamens are completely absent, and their inferior ovaries are much more developed than those of the tubular florets.The fruit forms only in the female ray flowers.The heterocarp achenes are sickle-shaped, curved and ringed.

Leaf, stem and root
The plant is an annual, seldom biennial.It grows to between 30 and 50 cm high, and has about 20 cm long tap root and numerous thin, secondary roots.The stem is erect, angular, downy and branched from the base up or higher.The alternate leaves are almost spatulate at the base, oblong to lanceolate above and are all tomentosae [5][6][7].

Traditional uses
In Europe, the leaves are considered resolvent and diaphoretic while the flowers are used as a stimulant, antispasmodic and emmenagogue [2].In England, the decoction of the flowers was used as a posset drink for the treatment of measles and smallpox, and the fresh juice as a remedy for jaundice, costiveness (constipation) and suppression of menstrual flow [8].In India, the florets are used in ointments for treating wounds, herpes, ulcers, frostbite, skin damage, scars and blood purification.The leaves, in infusion, are used for treating varicose veins externally [2,8].

Coumarins
The ethanol extract of the inflorescence of the C. officinalis reported to contain coumarins -scopoletin, umbelliferone and esculetin [29] .
Responsible for antitumor, antiinflammatory and antioedematous activities.

Amino acids
The ethanol extract of the flowers of the plant is reported to show the presence of 15 amino acids in free form: alanine, arginine, aspartic acid, aspargine, valine, histidine, glutamic acid, leucine, lysine, proline, serine, tyrosine, threonine, methionine and phenylalanine.Amino acid content of the leaves is about 5 %, stems 3.5 % and flowers 4.5 % [34].

Table 3: Structures and activities of some flavonoids
Structure Activity

Isorhamnetin
Responsible for antioxidant and wound healing activities

Responsible for antioxidant activities
Responsible for antioxidant activities

PHARMACOLOGICAL ACTIVITIES
Pharmacological studies have confirmed that C.officinalis exhibit a broad range of biological effects, some of which are very interesting for possible future development.

Anti-inflammatory and antioedematous activities
Ethyl acetate soluble fraction of the methanol extract of C. officinalis flowers exhibited the most potent inhibition (84 %) of 12-otetradecanoyl phorbol-13-acetate (TPA)induced inflammation (1 µg/ear) in mice with an ID 50 value of 0.05 -0.20 mg/ear compared with indomethacin as reference drug.Furthermore, activity-guided isolation showed that its activity was mainly due to oleananetype triterpene glycoside [18].A dose of 1200 µg/ear of an aqueous-ethanol extract showed 20 % inhibition in croton oil-induced mouse oedema.The activity was attributed to the presence of triterpenoids, the three most active compounds of which were the esters of faradiol-3-myristic acid, faradiol-3-palmitic acid and 4-taraxasterol [43,44].
Dichloromethane extract of the plant's flower heads inhibited croton oil-induced oedema, and further isolation showed that the esters of faradiol-myristic acid, faradiol-palmitic acid and ψ-taraxasterol had antioedematous activity with an oedema inhibition of nearly 50 % at a dose of 240 µg/cm 2 .Furthermore, when the doses of these two faradiol esters were doubled, oedema inhibition increased to 65 and 66 %, respectively, without any synergism between them [45].A cream containing calendula extract has been reported to be effective in dextran and burn oedemas as well as in acute lymphoedema in rats.Activity against lymphoedema was primarily attributed to enhancement of macrophage proteolytic activity [46].

Anti-HIV activity
Dichloromethane-methanol (1:1) extract of C. officinalis flowers exhibited potent anti-HIV activity in in vitro MTT/tetrazolium-based assay.This activity was attributed to inhibition of HIV1-RT at a concentration of 1,000 µg /mL as well as suppression of the HIVmediated fusion at 500 µg/mL [47].

Anticancer and lymphocyte activation dual activities
The ethyal acetate soluble fraction of the methanol extract of C. officinalis flowers has shown cytotoxic activity in vitro [18].Further activity-guided isolation of that fraction showed that the active compounds were: calenduloside F6'-O-n-butyl ester, which is active against leukaemia (MOLT-4 and RPMI 8226), colon cancer (HCC-2998) and melanoma (LOXIMVI, SK-MEL-5 and UACC-62)] cell lines with GI 50 values of 0.77-0.99µmole, except for leukaemia (CCRF-CEM, GI 50 = 23.1 µmole), renal cancer (AK-1, 17.2 µmole; UO-31, 12.7 µmole) and breast cancer (NCI/ADR-RES, >50 µmole)] cell lines; and calenduloside G6'-O-methyl ester, which is active against all the cancer cell lines mentioned above with GI 50 ≤ 20 µmole except for ovarian cancer (IGROVI, GI 50 = 20.1 µmole) and renal cancer (VO-31, 33.3 µmole) cell lines [18].Aqueous laser-activated calendula flower extract (LACE) showed potent in vitro inhibition of tumour cell proliferation when assayed against a wide variety of human and murine tumour cell lines.The inhibition ranged from 70 -100 % with an IC 50 concentration of 60 µg/mL.The mechanisms of the inhibition were identified as cell cycle arrest in G0/G1 phase and caspase-3 induced apoptosis.On the other hand, when LACE was assayed against human peripheral blood lymphocyte (PBLs) and human natural killer cell lines (NKL) it showed in vitro induction of proliferation and activation of these cells, mainly Blymphocytes, CD4 + , T lymphocytes and NKT lymphocyte [50].
Various extracts of the leaf, flower and whole plant have also been found to be cytotoxic to MRC5, HeP2, ascetic cells from Ehrlich carcinoma.The saponin rich fraction of these extracts displayed antitumoural activity in vivo in the Ehrlich mouse carcinoma model [51].

Antioxidant activity
A 70 % methanol extract of the plant was successively extracted with ether, chloroform, ethyl acetate and n-butanol leaving a residual aqueous extract which was assayed for antioxidant activity by liposomal lipid peroxidation-induced Fe 2+ and ascorbic acid.The ether, butanol and water extracts, containing flavonoids, showed antioxidant activity [54].Propylene glycol extracts of the petals and flower heads, assayed for antioxidant activity by lipid peroxidation, indicate that the extract of the petals was more potent than the flower head extract, based on analysis of plasma and urine malondialdehyde (MDA) and urine isoprostane inventrations (ipf2α-VI) [55].

Wound healing activity
The ethanol extract of the plant's flowers was investigated against experimentally induced thermal burns in rats.Among the various extract doses (20, 100, and 200 mg/kg of body weight), the 200 mg/kg dose showed significant improvement in healing of wounds as indicated by increase in collagenhydroxyproline and hexosamine contents.The level of acute phase proteins (heptaglobin, orosomycid and tissue damage marker enzymes -alkaline phosphatase), alanine and aspartate transaminase decreased significantly.The decrease in lipid peroxidation might be due to its antioxidant property [56].
The daily application of 2% calendula gel resulted in a greater number of wound healing due to its antimicrobial and antioxidant property [57].

Spasmolytic and spasmogenic dual activity
The aqueous-ethanol extract of C. officinalis flowers ,when assayed in rabbit jejunum, caused a dose-dependant (0.03 -3.0 mg/mL) relaxation of spontaneous and K + -induced contraction; further fractionation of the extract with dichloromethane showed inhibition of spontaneous contractions in a dose range of 0.01 -0.3 mg/mL.This is ten times more potent than the parent crude extract, and spasmolytic activity was found to be due to calcium channel blockade (CCB) [58].On the other hand, the aqueous fraction of the parent extract exhibited spasmogenic activity in a dose range of 1 -10 mg/mL [58].

Insecticidal activity
The acetone: methanol (2:1 v/v) extract of the flowers showed insecticidal activity when it was tested on milkweedbug [59].

Inhibition of heart rate
The aqueous extract was tested on the heart of male Wistar rats and found to inhibit heart rate contractility by up to 100 % at a dose of 0.3mg/L [60].

Genotoxic and antigenotoxic dual activities
The aqueous (AE), aqueous-ethanol (AEE), ethanol and chloroform extracts of C. officinalis flowers were evaluated to determine if they caused induction of unscheduled DNA synthesis (UDS) in rat liver culture and reversal of diethylnitrosamine (DEN)-induced UDS.In the UDS test in liver culture, DEN, at a level of 1.25 µmole, produced a maximum increase of 40% AEE extracts showed complete reversal of DEN effect at levels of around 50 ng/mL, and between 0.4 and 16 ng/mL, respectively.In the absence of DEN, these two polar extracts induced UDS at concentrations of 25 and 3.7 -100 µg /mL for AE and AEE, respectively, in rat liver cell culture.Thus these polar extracts (AE and AEE) at low concentrations (i.e., ng/mL range) showed antigenotoxic effect while at high concentrations (i.e., µg/mL range) they exhibited genotoxic effect [61].The propylene glycol extract of C. officinalis also showed antigenotoxic effect based on an evaluation in young growing pigs which involved the measurement of the excretion of lymphocyte DNA fragmentation and 24 h urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) [55].

Antiviral activity
A tincture of the flowers suppressed the replication of herpes simplex, influenza A2 and influenza APR-8 viruses in vitro [62].

TOXOCOLOGICAL STUDY
The hydroalcohol extract of C. officinalis flowers,, based on assessment in rats and mice, did not show acute toxicity following administration of an oral dose of up to 5.0 g/kg.It didn't show haematological alterations at doses of 0.025, 0.25, 0.5 and 1.0 g/kg.However, the biochemical parameters, blood urea nitrogen (BUN) and alanine transaminase (ALT), were elevated due to renal and liver overload [63].

CONTRAINDICATION
The extract was found to cause allergy in 9 patients out of 443 (2.03 %) when assessed by patch testing method [64].Therefore, it is advisable that the persons who have an established allergy to the Asteraceae (daisy) family should use it with caution [65,66] .

CONCLUSION
In this review, we have presented information on the botanical description, traditional uses, phytochemistry and pharmacology of C. officinalis Linn.(Asteraceae), a medicinal plant found in central and southern Europe, western Asia and the United States, amongst others.A variety of phytochemicals such as terpenoids, flavonoids, coumarins, quinones, volatile oil, carotenoids and others have been reported to be present in this plant.It exhibits several pharmacological activities such anti-HIV, anti-cancer (dual activity), antiinflammatory, hepatoprotective, spasmolytic and spasmogenic (dual activity), amongst others.It is potentially an important medicinal plant for mankind.

Fig 1 :
Fig 1: The leaf, stem and flower of C. officinalis while AE and

Table 2 :
Structures and activities of some terpenoids