Aplicación de la metabolómica

Paula Tyler Introduction

The Common Marigold or Pot Marigold, is formally known as

Calendula officinalis Linn. of the Asteraceae family (formerly

Compositae) (Gilman & Howe, 1999). This small and unassuming but brightly colored herb is also known as the Bride of the Sun, Mejorana or flaminquillo in Spanish, fior d’orgni in Italian, gauche-fer or souci des champs in French, goldblume or Ringelblume in German, and the golden flower of Mary (Basch et al., 2006). Native to Europe and parts of Asia, C.officinalis has been cultivated since the 12th _{century for}

medicinal use as an antiseptic, anti-inflammatory, cicatrizing agent, and antibacterial (Khalid & da Silva). C.officinalis is also used for culinary purposes, and for ethnoveterinary medicine. It contains many types of secondary metabolites, including terpenoids, flavonoids, carotenoids, volatile oils, quinones, lipids, and coumarins. Many of these compounds have potent pharmacological and biological activities that support its history of ethnomedicinal use.

Botanical Description

Calendula officinalis is a hardy cool season annual herb. Its

habit is round, with single or double showy white, orange, or yellow flowers (Figure 1). It grows 12-18 inches in height and width, and it is dense with simple ovate green leaves. It grows in full sun in acidic clay or sand and is known to attract butterflies (Gilman & Howe, 1999). C. officinalis is indigenous to Europe, and is cultivated in North America, Europe and the Balkans, and India (Khan et al., 2011).

Figure 1. Calendula officinalis. Note the characteristic yellow-orange color and the ovate leaves. (Image Source:

http://pharmacology.georgetown.edu/urbanherbs/calendula.htm) Traditional Uses

Ethnomedical Bangladesh

From an ethnomedicinal survey of the Kavirajes of the Kushtia District of Bangladesh, Calendula officinalis is known as Ganda. The leaf juice is applied topically for ear aches, skin infections, and insect bites (Rahmatullah et al., 2009).

India

For medicinal uses in India, Calendula officinalis cream is often combined with other herbs to treat hemorrhoids, burns, and abrasions. The florets in particular are made into ointments for wounds, herpes, ulcers, and other skin damage. Varicose veins are treated with an infusion of the leaves. Other ethnobotanical applications include the use of dried flowers as an insect repellent (Khalid & da Silva).

Italy

In a phytotherapy study conducted in the Peninsula Sorrentina of southern Italy, Calendula officinalis was a constituent of mixtures used for the treatment of unspecified varices. It is known locally as Calendula or sciure’e Sant’antonio (De Feo et al., 1992). C.officinalis teas are also used for eye infections, throat inflammation or pharyngitis, gingivostomatitis, and inflammatory skin conditions (Khalid & da Silva).

Portugal

Trás-os-Montes is a small area in Northern Portugal. Calendula

officinalis is known there as Calêndula, Mariana, or Maravilha.

The dry flowers are popularly used for problems with anxiety (nervousness and insomnia), the liver (jaundice), the eye (inflammation), digestion (ulcers), the skin (callus and warts), and incontinence (Neves et al., 2009).

Serbia

Calendula officinalis is locally known as Neven in the Kopaonik

Mountian area of central Serbia. An ointment of the plant is used for foot fungus, wounds, burns, and frostbite. Flowers are combined with boiled fat and then filtered after 24 hours. This

is used to treat oedema of the leg and painful veins. A tea is also prepared as a vermifuge (Jarić et al., 2007).

United Kingdom

In the UK, psoriasis, leprosy, measles, and smallpox were treated with a decoction of Calendula officinalis flowers, while the juice could be used for jaundice, constipation, and menstrual flow suppression (Khalid & da Silva).

United States of America

Similar to the Portuguese ethnomedicinal use, Calendula

officinalis was used in the US for ulcers, liver problems,

wounds, and conjunctivitis in the 19th _{century (Khalid & da}

Silva).

Ethnobotanical Culinary

As an alternative to saffron, C.officinalis is occasionally used in India as a colorant and for flavoring. (Khalid & da Silva).

Veterinary

In a survey of the Lower Mainland of Canada, Thompson/Okanagan Region and South Vancouver Island of British Columbia, livestock farmers reported using the flower oil of Calendula officinalis for wounds on their ruminants. The farmers specified that it is not generally used for deep wounds because the cicatrizing action of the herb can close the wound too quickly and seal infection inside. C.officinalis is also combined with Plantago spp., Urtica dioica and Symphytum

officinale for diarrhea and a tea is given orally for the relief of

a sore stomach (Lans et al., 2007).

Chemistry & Pharmacology

The flowers of Calendula officinalis contain various terpenoids, volatile oils, 15 amino acids, and fatty acids. Inflorescences contain carbohydrates, coumarins, and flavonoids. Terpenoids are found in the roots, while the leaves and stems contain carotenoids. Pollens and petals also contain carotenoids. Quinones are found in the leaves, cellular chloroplasts, and mitochondria. Seeds contain many neutral lipids in addition to various fatty acids. Loliolide is a main bitter constituent. Other compounds include calendulin, and n-paraffins (Muley et al., 2009). Table 1 contains an adapted summary of the chemical classes, names, and locations of these chemical compounds. Flavonoids and carotenoids are powerful antioxidants, while triterpenoids are efficacious anti-inflammatory agents. The structures of some of these terpenoids are demonstrated in Figure 2.

Biological Activity In Vivo Studies

Spasmolytic and Spasmogenic

The use of Calendula officinalis for gastrointestinal problems is less documented than some other uses, but there are spasmolytic and spasmogenic constituents in the flowers that could elucidate the plants effect on cramps and constipation. Fresh flowers were powdered and extracted in distilled water, dichloromethane, and ethyl acetate. The spasmolytic and spasmogenic activity was analyzed in 2 cm long segments of rabbit jejunum or guinea pig ileum, respectively. As a spasmolytic, C.officinalis inhibited the contraction of free Ca++

induced jejunum contraction in a dose-dependent fashion. A pretreatment dose of 0.3 mg/mL exhibited 80-90% inhibition against acetylcholine and histamine induced contraction in the

Figure 2. A summary of the structures of anti-

inflammatory triterpenoids in Calendula officinalis. (Image

Source: Neukirch et al., 2005)

guinea-pig ileum. The mechanism may be an interference of calcium release or influx.

The dichloromethane extract contained more spasmolytic constituents than the ethyl acetate or aqueous extracts. However, the aqueous extract had a dose-dependent spasmogenic effect on the guinea-pig ileum at 1-10 mg/mL but was partially blocked with pretreatment of atropine. This suggests that the spasmogenic mechanism as potentially similar to acetylcholine, which is important for regulation of peristalsis. By acting as a calcium channel blocker or cholinergic, C.officinalis is capable of either relaxant or stimulant actions (Bashir et al ., 2006).

Chemical

Class Compounds Plant Part Activities

Terpenoids 1) Sitosterols, stigmasterols, diol diesters, taraxasterol monoesters, erythrodiol, brien, ursadiol, faradiol esters, arnidiol esters, calenduladiol esters, oleanolic acid saponins, calendulosides, calendulaglycosides, glucuronides, and cornulacic acid

2) Oleanolic acid glucosides

1) Flowers 2) Roots

Antioedematous Anti-

inflammatory

Flavanoids Quercetin, isorhamnetin, isoquercitin, narcissin, calendoflaside,

calendoflavoside,

calendoflavobioside, rutin, isoquercetirin,

neoheperididosides, and isohamnetin and quercetin rutinosides

Inflorescence Proteolytic Enzyme Inhibition Antiviral

Coumarins Scoplotetin, umbelliferone, esculetin Inflorescence Quinones 1) Plastoquinone, phylloquinone

2) α-tocepherol 3) Ubiquinone, phylloquinone, α- tocepherol 1) Leaves 2) Chloropl asts 3) Mitocho ndria Volatile Oil Monoterpenes and sesquiterpenes: α-thujene, α-

pinene, limonene, 1,8- cineol, geraniol,

Essential oil: α-cadinene, α- cadinol, tmuurolol, lionene, and 1,8-cineol

Flowers Allergen

Carotenoids 1) neoxanthins, violaxanthins, luteoxanthins, auroxanthin, favoxanthin, luteins, cryptoxanthins, lycopene, α-carotene, β- carotene 2) neoxanthins, 1) Pollens and Petals 2) Leaves and Stems Anti- inflammatory Choleretic- Cholagogue Antioxidant Increase Tumor Latency violaxanthins, luteoxanthin, antheraxanthin, mutatoxanthins, luteins, cryptoxanthins, and β- carotene

Amino Acids alanine, arginine, aspartic acid, asparigine, valine, histidine, glutamic acid, leucine, lysine, proline, serine, tyrosine, threonine, methionine, and phenylanlanine Flowers Carbohydrat es Polysaccharides and monosaccharides Inflorescence Lipids 1) Neutral lipids: phospholipids, glycolipids

and Fatty acids: conjugated trienic acid and dimorphecolic acid 2) Fatty acids: lauric,

myristic, palmitic, stearic, oleic, and linoleic monols, sterol esters, 3-

monoestersm and 3- monoester diols 3) oxygenated fatty acid

1) seeds 2) flowers 3) seed oil

Other Loliolide Calendulin n-paraffins

Bitter constituents Table 1: Summary of Phytochemical Constituents of Calendula officinalis L. (Asteraceae) and examples of known bioactivity

(adapted from Muley et al., 2009).

Antidiabetic & Antihyperlipidaemic

The search for natural products efficacious in the treatment of diabetes mellitus is worth pursuing because synthetic and semi-synthetic drug products can have undesirable side effects. Chakraborthy et al. (2011) examined the ability of

Calendula officinalis to affect the metabolism of glucose and

lipid peroxidation in rats with induced diabetes. The study

used an 8.7 % extract of the leaves in ethanol and water. Alloxan and glucose were administered intraperitoneally to induce diabetes in rats, and 36 rats with a resulting blood glucose range of 200-270 mg/dL after two weeks were given either 2 mL saline as a control, 25 mg/kg, 50 mg/kg, or 100 mg/kg of C.officinalis, or 6 U/kg of insulin orally for 42 days. Blood glucose and urine sugar were measured at 2 weeks, 4 weeks, and 6 weeks. The response of blood glucose and urine sugar to C.officinalis extract was determined to be dose dependent. The 100 mg/kg dose actually restored the blood glucose/urine sugar to normal levels, while the 25 and 50 mg/kg dose still significantly lowered these indicators. There was an overall significant increase in haemoglobin and body weight when given C.officinalis. The lipid levels in blood serum were also lowered. The proposed mechanism of the extract is the activation of increased insulin secretion from the pancreas. Because the effects of the extract are so similar to that of administered insulin, the potential of C.officinalis for antidiabetic and antihyperlipidaemic use at 100 mg/kg body weight (BW) should be explored further for human application (Chakraborthy et al., 2011).

Hepatoprotective

Potential hepatoprotective properties of Calendula officinalis were examined using induced hepatic toxicosis in rats. Carbon tetrachloride (CCl4) produces the free radical CCl3, which

affects lipid peroxidation and enzyme activity. CCl4 was given

to induce the toxicosis, and the animals were treated with an ethanol extract of powdered flowers every 24 hours for 7 days beginning 30 minutes after intoxication. C.officinalis showed a positive effect by decreasing hepatocytolysis by 28.5% and reducing enzyme and steatosis changes. The carotenoids, flavones, saponins, and phenylpropanoids are believed to be

responsible for the anti-inflammatory and choleretic- cholagogue activity (Rusu et al., 2005).

Antioxidant

Oxidative stress, resulting from an excess of free radicals and lack of antioxidant action can result in damage to lipids, proteins, and DNA. The carotenoid and polyphenol content of

Calendula officinalis is considered high and indicative of

antioxidant activity, so Frankič et al. (2009) explored the effectiveness of C.officinalis extract for protection against DNA damage and lipid peroxidation in pigs. The extract was prepared from flower tops or petals in propylene glycol and experimental groups received either 3mL/day of petal extract or 3 mL/day of flower top extract in the feed. Oxidative DNA damage was induced by high polyunsaturated fatty acids (PUFA) in the feed, and both C.officinalis extracts significantly decreased the amount of DNA damage. High PUFA content also raises the marker of lipid peroxidation in urine, but the amount of C.officinalis extract was not sufficient enough to reduce the lipid peroxidation in this study. In summary, these therapeutic amounts of C. officinalis are considered efficacious enough in antioxidant activity for the protection of DNA, but are not sufficient for inhibiting lipid peroxidation (Frankič et al., 2009).

Anticancer

Calendula officinalis contains lutein, which is a potent

carotenoid antioxidant. High levels of lutein have previously been shown to correlate with survival rates and the expression of estrogen receptors in breast cancer cells. In order to examine the potential of low dietary lutein as an inhibitor of mammary cancer cells, a murine mammary tumor cell culture was prepared and infused into female mice. After

the amount required to induce 65% tumor incidence was determined, 30 mice were given a semi-synthetic diet with 0.002, 0.02, and 0.4% extract from C.officinalis and inoculated with the tumor cells after two weeks. At 70 days post- inoculation the solid tumors were excised and lipid peroxidation activity was determined. Mice fed 0.02 and 0.4% lutein had a tumor incidence of only 20-37% compared to 70% in non-supplemented mice, and final tumor incidence was greater at day 70 for the untreated mice. Mice fed 0.002% had a tumor latency of about 50 days, while the tumor latency was delayed by 15 days with other concentrations. Lipid peroxidation was not dose dependent on lutein. Though lutein from C.officinalis did not prevent tumor growth, it may prevent the initial establishment of tumor cells as indicated by the increased tumor latency. The proposed mechanism of action is the antioxidant activity of lutein by quenching singlet oxygen and possibly additional immunomodulation, cell-cell communication, and prostaglandin production (Park, Chew, & Wong, 1998).

Antioedemous

Three of the most powerful compounds in Calendula officinalis include the tripterpenoid alcohols faradiol-3-myristic acid ester, faradiol-3-palmitic acid ester and ᵠ- taraxasterol. Their efficacy as antioedemic agents was investigated by Zitterl et al. (1997). The compounds were extracted from the powdered flower heads with dichloromethane. The irritant, Croton oil, was applied to the right inner ear surface of mice and the left ear was left untreated for comparison. Experimental mice received a mixture of Faradiol monoesters, Faradiol-3- myristic acid ester, Faradiol-3-palmitic acid ester, ᵠ- taraxasterol, and Faradiol, or the control Indomethacin while control groups only received the irritant. After 6 hours, plugs

used to measure the size of the ear canal in both ears of each animal were removed and the difference in plug weight indicated the amount of swelling. Faradiol-3-myristic acid ester and Faradiol-3-palmitic acid ester exhibited 50% oedema inhibition at 240 µg/cm2 _{and 65-66% at 480µg/cm}2_.

On a molar basis, 21 µmol/cm2 _{of ᵠ- taraxasterol is needed to}

achieve the same effect. It was proposed that the limited activity at higher doses may be due to the difficulty of lipophilic compounds moving through the epidermis. It was concluded that Faradiol was as effective as Indomethacin and more active than the esters or ᵠ- taraxasterol (Zitterl-Eglseer et al., 1997).

Anti-inflammatory

Reducing inflammation is perhaps the best known activity of

Calendual officinalis and most common ethnomedicinal

application, so Preethi et al (2009) studied this particular property in a multi-pronged study. Similar to the antioedematous activity, the combination of caratenoids, flavanoids, and triterpenoids in C. officinalis was found to mediate acute and chronic inflammation in mice by cytokine and macrophage inhibition, and free radical scavenging.

Flower tops were extracted with ethyl alcohol to a yield of 1.1 g/100 mL, dried, and dissolved in distilled water for experimental use, and 8 groups of 6 mice received an injection of carrageenan and Dextran in the right paw to model acute inflammation. The control groups orally received only carageenan, distilled water, Dextran, or Diclofenac. The experimental groups orally received either 250 or 500 mg/kg BW of Calendula officinalis extract. Caliper measurements of the paw thickness were taken before and after injection and every hour. Conversely, Formalin was used to model chronic inflammation in 4 groups of 6 mice. These groups received no

treatment, 250 or 500 mg/kg BW of extract, or Diclofenac 1 hour before an injection of Formalin and for 6 days afterward. Calipers were used to measure the paw every day. The extract significantly reduced acute and chronic edema with 50.6- 65.9% and 62.3% inhibition, respectively.

The effect of Calendula officinalis on TNF-α, a tumor necrosis factor from macrophages, was also examined in this study. Macrophages were induced by sodium caesinate in experimental groups of mice. The mice were treated with the extract alone at 100 or 250 mg/kg BW or with lipopolysaccharide (LPS) + extract at 100 or 250 mg/kg BW for 5 days. LPS was injected on the 5th _{day and after 6 hours}

the macrophages were collected for the TNF-α activity assessment. Treatment with the extract resulted in minimal macrophage cytotoxicity and normal cell growth.

The extract’s effect on proinflammatory cytokines was determined by treatment of mice with LPS, or 50, 100, or 250 mg/kg BW LPS and Calendula officinalis. The LPS was administered after 5 days, and the levels of cytokines were determined after 6 hours. The cytokinase level in response to inflammation was also inhibited by C.officinalis.

Inflammation was also induced by LPS after 5 days of receiving 100 or 250 mg/kg BW of extract to determine the expression of the cyclooxygenase-2 gene. RNA was isolated from the spleen, and cDNA was prepared by RT-PCR and amplified. Band intensity of the Cox-2 gene was lower in

C.officinalis treated animals, indicating expression inhibition.

In summary, the proposed mechanism for the anti- inflammatory action of the extract is the modulation of cytokines and Cox-2 gene inhibition in addition to antioxidant action against activated macrophages (Preethi, Kuttan, & Kuttan, 2009).

Cutaneous Wound Healing

Calendula officinalis has long been used in topical applications

for wound healing because of its anti-inflammatory, cicatrizing action, and anti-microbial properties. The effect of a gel containing C. officinalis on collagen production and wound healing in rats was studied by Naeini et al. (2012). The oily product was extracted from fresh flower tops with ethyl alcohol, dried, and a 5%, 7%, and 10% gel were created. The rats received a square 2x2 cm surgical skin incision under anesthesia, and the gel was applied daily for 14 days. Control rats received no gel or a gel base placebo, and experimental rats received 5% gel, 7% gel, or 10% gel and they were observed daily. The wounds were investigated by biopsy at days 14, 21, and 45. At day 21, the collagen and hydroxyproline content were higher for the 7% and 10% groups, but on days 14 and 45 the 7% group was the highest. It was concluded from statistical evaluation that the 7% gel was more effective that the other concentrations because it showed significant collagen synthesis. Furthermore, the

C.officinalis gel was most effective at day 14 (Naeini et al.,

2012).

In Vitro Studies

Antimicrobial& Antifungal

One aspect of Calendula officinalis wound healing action may be the antimicrobial and antifungal properties of the plant. These activities were studied by Efstratiou et al (2012). Petals from the flowers were isolated, dried, and extracted in methanol or ethanol. Each extract was tested against Bacillus

subtilis, B. cereus, B. pumilis, Pseudomonas aeruginosa, three

strains of Escherichia coli and ampicillin-resistant E.coli,

Staphylococcus aureus, Klebsiella aerogenes, Enterococcus faecalis, two strains of Candida albicans, C. krusei, C. glabrata, 139 | M e d i c i n a l P l a n t M o n o g r a p h s

C. parapsilos, Aspergillus flavus, A. fumigates, A. niger, and Exophiala dermatiditis. The extracts were dissolved to 300

mg/mL in their respective solvents, and 15 µL were used to soak paper discs. Bacterial controls were Ciprofloxacin and solvents. 10 mg/mL extracts were used for the fungal test and the controls were Fluconazole and the solvents. The antimicrobial activity, indicated by growth inhibition, was observed by disc diffusion. C.officinalis was comparable against both Gram-positive and Gram-negative samples, and methanol was generally more effective. However, S. aureus

and E. faecalis were more susceptible to the ethanol extract.

Both methanol and ethanol extracts were comparable in antifungal activity to Flucanazole (Efstratiou et al., 2012).

Immunomodulatory

It is possible that Calendula officinalis regulates inflammation by regulating the immune response on a cellular level. To test this hypothesis, Amirghofran et al (2000) prepared ethanol extracts of C.officinalis and other herbs. Human lymphocytes and thymocytes were isolated and assayed with the extract. Proliferation was significantly inhibited by C.officinalis and dose-dependent. The potential mechanism is through interaction with growth factors or cell surface molecules (Amirghofran, Azadbakht, & Karimi, 2000).

Antiviral

Calendula officinalis was investigated for antiviral activity by

Kalvatchev et al (1997). The dried flowers were extracted as organic and aqueous stock solutions. The aqueous extract did not show inhibition of HIV-1 replication in infected cells, but the organic extract was an effective inhibitor at 10-30 µg/mL. Again, the aqueous extract was less effective than the organic